Genetic diagnosis for qt prolongation related adverse drug reactions

ABSTRACT

The specification is directed to a method of diagnosing whether a subject is predisposed to an adverse reaction to one or more pharmaceutical agents which may induce a prolonged QT interval or acquired LQTS in that individual. The diagnosis is genetic analysis of at least two polymorphisms or mutations which the individual may have, which are associated with an increased risk for prolonged QT intervals or Torsades de Pointes (TdP). Genetic screening for determining the predisposition of prolonged QT intervals induced by a pharmaceutical agent is performed by identifying genetic polymorphisms or mutations located in at least two classes of genes, wherein the genes are (1) LQT genes, (2) altered sensitivity genes (e.g., MiRP1) or (3) increased exposure genes (e.g., MDR genes or P450 cytochrome genes). The specification is also directed to compositions and kits for determining such predispositions to adverse drug reactions.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of priority from U.S. ProvisionalPatent Application Serial No. 60/196,916, filed Apr. 13, 2000, theentirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to methods of determining a predispositionfor QT interval prolongation in a subject after the administration of apharmaceutical agent or agents. Compositions and kits for determiningsaid predispositions to the QT interval prolongation are also described.

BACKGROUND OF THE INVENTION

[0003] The invention relates to a method of screening a subject for apredisposition to an adverse drug reaction involving prolonged QTintervals. The genetic screening of patients for said predispositionfocuses on genes associated with QT interval prolongation, including LQTgenes, P-glycoprotein membrane pump proteins (P-gp), multidrugresistance genes and cytochrome P450-mediated drug metabolism genes.

[0004] I. LQT and Cytochrome P450 Genes and Polymorphisms

[0005] 1. LQT Genes

[0006] Genes associated with long QT (LQT) syndrome (LQTS) includeKVLQT1 (LQT1), HERG (LQT2), SCN5A (LQT3) and MinK (LQT5). A fifth genelocus exists on human chromosome 4 (e.g., LQT4). Recently, a sixth gene(LQT6) has been identified (Wang et al., Ann. Med. 30: 58-65 (1998)).All but LQT3 encode cardiac potassium ion (K⁺) channel proteins; LQT3encodes a cardiac sodium ion (Na⁺) channel protein (Vincent, Annu. Rev.Med. 49: 263-74 (1998)). At least 180 mutations have been identifiedamong these genes (Abbott et al., Cell 97: 175-87 (1999); Vincent. Annu.Rev. Med. 49: 263-74 (1998); Curran et al., Cell 80: 795-803 (1995);Berthet et al., Circulation 99: 1464-70 (1999); Dausse et al., J. Mol.Cell Cardiol. 28: 1609-15 (1996); Chen et al., J Biol. Chem. 274:10113-8 (1999); and Sanguinetti et al., Proc. Natl. Acad. Sci. U.S.A.93: 2208-12 (1996)). Some of these mutations cause altered ion channelfunction resulting in non-drug induced prolonged QT intervals and apropensity for Torsades de Pointes (TdP) (See, e.g., Berthet et al.,Circulation99: 1464-70 (1999)). Accordingly, genetic screening can beperformed on subjects suspected of having long QT syndrome, as well asother patients (see, e.g., Satler et al., Hum. Genet. 102: 265-72(1998)). Larson et al., Hum. Mutat. 13: 318-27 (1999) reported ahigh-throughout single strand polymorphism (SSCP) analysis for detectingpoint mutations associated with LQTS.

[0007] U.S. Pat. No. 5,599,673 claims two (e.g., HERG and SCN5A) of thesix LQT genes. Two HERG-related genes have also been claimed (U.S. Pat.No. 5,986,081). International PCT Application WO 97/23598 describes amethod of assessing a patient's risk for long QT syndrome (LQTS) byscreening for genetic mutations in the MinK gene. However, these patentsdo not disclose methods of diagnosing a patient's predisposition to anadverse drug reaction involving elongation of the QT interval due tomutations in any of the LQT genes.

[0008] Drugs have been identified that cause QT interval prolongation,and thereby adverse drug reactions. Certain antihistamines, such asterfenadine (e.g., Seldane®) and astemizole (e.g., Hismanal®),.reportedly block potassium channels (Woosley, Annu. Rev. Pharmacol.Toxicol. 36: 233-52 (1996)) and inhibit the HERG protein, and therebywere postulated to induce Torsades de Pointes (Wang et al., 1998). Allantiarrhythmic drugs that lengthen repolarization reportedly can causeTorsades de Pointes (Drici et al., Circulation94: 1471-4 (1996)).Additional non-cardiac and cardiac drugs capable of inducing QTprolongation including many that were identified by the inventor werereleased on Mar. 27, 1998 at the following web site: www.qtdrugs.org.However, Wei et al., Circulation 92:1-125 (1995) could not identify HERGor SCN5A gene mutations that were linked to acquired LQTS in patientstreated with an anti-arrhythmic agent. To the best knowledge of theinventor, no one has described diagnosing a predisposition towards anadverse drug or drug-drug reaction which causes QT interval elongationby screening patients for one or more polymorphisms in one or more LQTgenes.

[0009] 1. Cytochrome P450 Genes

[0010] The cytochrome P450 enzymes have also been linked to adverse drugreactions. CYP2D6 was the first cytochrome P450 isoform found to begenetically polymorphic in its distribution (Eichelbaum et al., Eur. J.Clin. Pharmacol. 16: 183-7 (1979); and Mahgoub et al., Lancet 2: 584-6(1977)), and it is now clear that this enzyme metabolizes a large numberof drugs (Inaba et al., Can. J. Physiol. Pharmacol. 73: 331-8 (1995);and Buchert et al., Pharmacogenetics 2: 2-11 (1992)). At least 30mutations exist which alter the activity or specificity of CYP2D6(Jordan et al., Endocr. Rev. 20: 253-78 (1999)). These include allelesthat contain single point mutations resulting in no activity (e.g.,CYP2D6*4), alleles in which the CYP2D6 gene has been deleted (e.g.,CYP2D6*5) and alleles in which it has been duplicated (e.g., CYP2D6*2_n)(Aklillu et al., J. Pharmacol. Exp. Ther. 278: 441-6 (1996)).

[0011] There are numerous cytochrome P450 genes which are involved inthe metabolism of drugs and drug metabolites. Several of them includeCYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7.Allelic variations exist amongst these genes. Certain of these allelicvariations combine to produce a poor metabolizer phenotype in 7% ofCaucasians, but smaller percentages of Africans and Asians and the“ultrarapid” phenotype in ˜5% of Caucasian and up to 30% Africans. Asethnic-specific alleles for both Asians (Yokoi et al., Pharm. Res. 15:517-24 (1998)) and Africans (Aklillu et al., J. Pharmacol. Exp. Ther.278: 441-6 (1996); and Oscarson et al., Mol. Pharmacol. 52: 1034-40(1997)) have been identified, that may alter the mean activity of theenzymes in these populations (see Table 1 below), it is also importantto test for these alleles in studies of the relationship betweengenotype and phenotype. TABLE 1 Chromosome Distribution of CytochromeP450 Gene Chr. Chr. 10 Chr. 10 Chr. 22 15 Polymorphic PolymorphicPolymorphic Chr. 10 Chr. 7 3-5% 1-3% 5-10% Caucasian Caucasian CaucasianPMs PMs PMs 15-20% Asian PMs

[0012] In fact, due to the metabolic differences, methods have beenreported which identify a drug which interacts with the CYP2C19 geneproduct, S-mephenytoin 4′-hydroxylase (U.S. Pat. No. 5,786,191).

[0013] Methods for detecting the presence or absence of mutations incertain of the cytochrome P450 genes have been described. For example,U.S. Pat. No. 5,891,633 relates to a method of identifying mutations inthe cytochrome P450 genes CYP2C9 and CYP2A6.

[0014] International PCT Application WO 95/30772 reportedly describes aCYP2D6 gene polymorphisms involving a 9 bp insertion in exon 9, which isassociated with a slower than normal rate of drug metabolism inindividuals bearing it and may be therefore useful diagnostically. PCRprimers have been described for detecting mutations in drug metabolismenzymes, including detection of the debrisoquine polymorphism,mephenytoin polymorphism and the acetylation polymorphism (U.S. Pat.Nos. 5,648,484 and 5,844,1 08). Additional mutations have beenidentified in CYP2D6 bufuralol-1′-hydroxylase, including mutations atpositions 271, 281, 294, and 506 which result in metabolizer/poormetabolizer phenotypes as described in International PCT Application WO91/10745 and U.S. Pat. No. 5,981,174.

[0015] Japanese Patent No. 8168400 provides a method of determiningmutations in exons 6 and 7 of the CYP2C19 gene. Japanese Patent No.10014585 describes primers and methods of detecting a mutation in exon 5of CYP2C19, which is related to the abnormal metabolism of diazepam,imipramine, omeprazole and propranolol. U.S. Pat. No. 5,912,120 claims amethod of diagnosing a patient having a deficiency in S-mephenytoin4′-hydroxylase activity by detecting polymorphisms at nucleotides 681 or636.

[0016] U.S. Pat. No. 5,719,026 provides methods and primers fordetecting a polymorphisms in CYP1A2 and assessing the changes in thedrug activity of theophylline associated with those polymorphisms.

[0017] Japanese Patent No. 10286090 reportedly describes methods andprimers to detect mutations in CYP2E1. These mutations are reported asbeing useful for determining the safety margin for drug administrationfor the treatment or related diseases.

[0018] Despite these teachings and to the best of the inventor'sknowledge, no one has described or suggested that a combination ofpolymorphisms in LQT and cytochrome P450 genes can induce acquired LQTSin a subject in response to the administration of a drug or drugs.

[0019] C. P-glycoprotein Pump Genes

[0020] P-Glycoprotein Pump (P-gp) in the development of drug-resistanttumor cells has been extensively studied (Lo et al., J. Clin. Pharmacol.39: 995-1005 (1999)). P-gp is an ATP-dependent drug pump that extrudes abroad range of cytotoxic agents from the cells end is encoded by a genecalled MDR-1, for multidrug resistance (Loo et al., Biochem. Cell. Biol.77: 11-23 (1999); and Robert, Eur. J. Clin. Invest. 29: 536-45 (1999)).The human P-gp sequence has been described by Chen et al., Cell 47:381-9 (1986) and has the GenBank Accession No. M14758.

[0021] Its physiological role may be to protect the body from endogenousand exogenous cytotoxic agents. The protein has clinical importancebecause it contributes to the phenomenon of multidrug resistance duringchemotherapy (Loo et al., 1999) and the development of simultaneousresistance to multiple cytotoxic drugs in cancer cells (Anbudkar et al.,Annu. Rev. Pharmacol. Toxicol. 39: 361-98 (1999)). Specifically, theover expression of this membrane pump appears to extrude manyxenobiotics out of the tumor cells (Robert., 1999). However,considerable controversy remains about the mechanism of action of thisefflux pump and its function in normal cells (Ambudkar et al., 1999).

[0022] Multidrug resistance (MDR) can be diagnosed in tumors usingmolecular biology techniques (e.g., gene expression at the mRNA level),by immunological techniques (e.g., quantification of the P-glycoproteinitself) or by functional approaches (e.g., measuring dye exclusion)(Robert, 1999).

[0023] Drugs have been developed which reverse or modulate MDR. Forexample, PSC-833 is a non-immunosuppressive cyclosporin derivative thatpotently and specifically inhibits P-gp (Atadja et al., CancerMetastasis Rev. 17: 163-8 (1998)). Also, compounds have been identifiedwhich increase or modulate the bioavailability of pharmaceuticalcompounds. See, e.g., U.S. Pat. Nos. 6,004,927; 5,962,522; 5,916,566;5,716,928; 5,665,386; and 5,567,592. P-gp activity has been altered byexpression of antisense nucleotides specific to MDR-1 (U.S. Pat. No.6,001,991). Methods and assays have also been developed which assesswhether multidrug resistance has been reversed (U.S. Pat. No.5,403,574).

[0024] Mutations have also been identified which alter an agentsinteraction with P-gp. For instance U.S. Pat. No. 5,830,697 disclosessingle and double mutations (Phe335 and/or 336) which alters thespectrum of cross-reactivity to cytotoxins and resistance to modulationby cyclosporins. Another mutation at V185G in P-gp confers increasedresistence to colchicine (U.S. Pat. No. 5,830,697). P-gp sensitivity tovinblastine, colchicine, VP16 and adriamycin, common chemotherapeuticagents, was up- and down-regulated by altering ⁶¹His to another aminoacid residue (Taguchi et al., Biochemistry 36: 8883-9 (1997)). Moreover,different drugs interact differently with P-gp and mutated forms ofP-gp, such that one mutation may influence the activity of one drug andnot another (See, e.g., Chen et al., J. Biol. Chem. 272: 5974-82 (1997);Bakos et al., Biochem. J. 323: 777-83 (1997); and Gros et al., Proc.Natl. Acad. Sci. USA 88: 7289-93 (1991)). However, despite theinformation regarding the influence such mutations may have on drugactivity, no association has been made linking P-gp by itself or incombination with another protein in influencing QT intervals or inducingTorsades de Pointes.

[0025] II. Nucleic Acid Hybridization

[0026] The capacity of a nucleic acid “probe” molecule to hybridize(i.e., base pair) to a complementary nucleic acid “target” moleculeforms the cornerstone for a wide array of diagnostic and therapeuticprocedures Hybridization assays are extensively used in molecularbiology and medicine. Methods of performing such hybridization reactionsare disclosed by, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), Haymes et al., Nucleic Acid Hybridization: APractical Approach (IRL Press, Washington, D.C. (1985)) and Keller etal., DNA Probes (²nd Ed., Stockton Press, New York (1993)).

[0027] Many hybridization assays require the immobilization of onecomponent to a solid support. Nagata et al., FEBS Letters 183: 379-82(1985) described a method for quantifying DNA which involved bindingunknown amounts of cloned DNA to microtiter wells in the presence of 0.1M MgCl₂. A complementary biotinylated probe was then hybridized to theDNA in each well and the bound probe measured colorimetrically. Dahlenet al., Mol. Cell. Probes 1: 159-168 (1987) have discussed sandwichhybridization in microtiter wells using cloned capture DNA adsorbed tothe wells. An assay for detecting HIV-1 DNA using PCR amplification andcapture hybridization in microliter wells also has been reported (Kelleret al., J. Clin. Microbial. 29: 638-41 (1991)). The NaCl-mediatedbinding of oligomers to polystyrene wells has been discussed by Cros etal. (French Patent No.2,663,040) and by Nikiforov et al., PCR MethodsApplic. 3: 285-291 (1994). A cationic detergent-mediated binding ofoligomers to polystyrene wells has been described by Nikiforov et al.,Nucleic Acids Res. 22: 4167-75 (1994).

[0028] III. Analysis of Single Nucleotide DNA Polymorphisms

[0029] Many genetic diseases and traits (i.e. hemophilia, sickle-cellanemia, cystic fibrosis, etc.) reflect the consequences of mutationsthat have arisen in the genomes of some members of a species throughmutation or evolution (Gusella, Ann. Rev. Biochem. 55: 831-54 (1986)).In some cases, such polymorphisms are linked to a genetic locusresponsible for the disease or trait; in other cases, the polymorphismsare the determinative characteristic of the condition.

[0030] Single nucleotide polymorphisms (SNPs) differ significantly fromthe variable nucleotide type polymorphisms (VNTRs), that arise fromspontaneous tandem duplications of di- or tri-nucleotide repeated motifsof nucleotides (Weber, U.S. Pat. No. 5,075,217; Armour et al., FEBSLett. 307: 113-5 (1992); Horn et al., PCT Application No. WO 91/14003;Moore et al., Genomics 10: 654-60 (1991); Hillel et al., Genet. 124:783-9 (1990)), and from the restriction fragment length polymorphisms(“RFLPs”) that comprise variations which alter the lengths of thefragments that are generated by restriction endonuclease cleavage (e.g.,Fischer et al., (PCT Application No. WO 90/13668); and Uhlen (PCTApplication No. WO 90/11369)).

[0031] Because SNPs constitute sites of variation flanked by regions ofinvariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation; it is unnecessary to determine a complete genesequence for each patient. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

[0032] Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses amethod for determining the identity of the nucleotide present at aparticular polymorphic site that employs a specializedexonuclease-resistant nucleotide derivative.

[0033] Cohen et al. (French Patent 2,650,840; and PCT Application No. WO91/02087) discuss a solution-based method for determining the identityof the nucleotide of a polymorphic site. As in the Mundy method of U.S.Pat. No. 4,656,127, a primer is employed that is complementary toallelic sequences immediately 3′ to a polymorphic site.

[0034] Additional SNP detection methods include the Genetic Bit Analysismethod described by Goelet et al. (PCT Application No. 92/15712). Themethod of Goelet et al. uses mixtures of labeled terminators and aprimer that is complementary to the sequence 3′ to a polymorphic site.

[0035] Cheesman (U.S. Pat. No. 5,302.509) describes a method forsequencing a single stranded DNA molecule using fluorescently labeled3′-blocked nucleotide triphosphates. An apparatus for the separation,concentration and detection of a DNA molecule in a liquid sample hasbeen described by Ritterband et al. (PCT Patent Application No. WO95/17676).

[0036] Several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Prezant et al.,Hum. Mutat. 1: 1 59-64 (1992); Ugozzoli et al., GATA 9: 107-12 (1992);and Nyren et al., Anal. Biochem. 208: 171-5 (1993)).

[0037] IV. Methods of Immobilization Nucleic Acids to a Solid-Phase

[0038] Several of the above-described methods involve procedures inwhich one or more of the nucleic acid reactants are immobilized to asolid support. Currently, 96-well polystyrene plates are widely used insolid-phase immunoassays. PCR product detection methods that use platesas a solid support and DNA chips have been described. The microtiterplate method requires the immobilization of a suitable oligonucleotideprobe into the microtiter wells, followed by the capture of the PCRproduct by hybridization and colorimetric detection of a suitablehapten.

[0039] Covalent disulfide bonds have been previously used to immobilizeboth proteins and oligonucleotides. Chu et al. (Nucl. Acids Res. 16:3671-91 (1988)) discloses a method for coupling oligonucleotides tonucleic acids or proteins via cleavable disulfide bonds.

[0040] Gentalen et al., Nucl. Acids Res. 27: 1485-91 (1999) describe acooperative hybridization method lo establish physical linkage betweentwo loci on a DNA strand by using hybridization to a new type ofhigh-density oligonucleotide array. This same method can be used todetermine SNP haplotypes.

[0041] Yershov et al., Proc. Natl. Acad. Sci. USA 93: 4913-8 (1996)describe an oligonucleotide microchip which has been used to detectbeta-thalassemia mutations in patients by hybridizing PCR-amplified DNAwith the microchips. This technology was suggested for large scalediagnostics in gene polymorphism studies.

[0042] Guo et al., Nucl Acids Res. 22: 5456-65 (1994) describe a simplemethod for the analysis of genetic polymorphisms allele-specificoligonucleotide raised bound to glass supports. This method wasdemonstrated in parallel analysis of 5 point mutations from exon 4 ofthe human tyrosinase gene.

[0043] More recently. Gilles et al., Nat. Biotechnol . 17: 365-70 (1999)have described a rapid assay for SNP detection utilizing electronicscircuitry on silicon Microchips. Holloway et al., Hum. Mutat. 14: 340-7(1999) also compares methods for high-thoughput SNP typing using TaqMan®liquid phase hybridization, PCR-SSOP or, ARMS-microplate array diagonalgel electrophoresis (MADGE).

[0044] As the world population ages and new drugs are identified, moreand more patients will administer one or more pharmaceuticalcompositions, such that an individual drug or drugs combination cancause adverse drug reactions. Therefore, not withstanding what has beenpreviously reported in the literature, the inventor herein describesmethods and compositions for diagnosing drug interactions which involveat least one mutation in a LQT gene. Additional mutations may exist incertain cytochrome 450 genes and P-glycoprotein pumps, which work inconcert with a LQT gene mutation or other ion channel (e.g., K⁺ or Na⁺)gene polymorphisms to produce an adverse drug or drug-drug reaction. Thespecification also discloses kits and compositions for diagnosing asubject's predisposition to QT interval elongation in response to theadministration of one or more pharmaceutical agents.

SUMMARY OF THE INVENTION

[0045] It is an object of the invention to provide novel and improvedmethods for determining whether a subject has a predisposition for QTinterval elongation or Torsades de Pointes due to one or morepharmaceutical agents. The methods comprise the step of screening abiological sample from the subject through a nucleic acid array, whereinsaid nucleic acid array contains probes for at least two geneticmutations or polymorphisms. These two genetic mutations or polymorphismsare located in two or more of the group of genes consisting of (1) LQTgenes, (2) altered sensitivity genes, and (3) increased exposure genes.Preferred genes include LQT genes and MDR genes (e.g., MDR-1). Thenucleic acid array can be in the form of a chip, a microchip, a bead ora microsphere. The LQT gene which may contain a polymorphism whichinduces QT internal elongation include LQT1, LQT2, LQT3, LQT4, LQT5 andLQT6.

[0046] The method may further comprise screening both LQT and increasedexposure gene (e.g., cytochrome P450 genes) mutations and polymorphism.The P450 cytochrome isoforms which may contain a mutation which canresult in excessive accumulation of drugs and thereby induce QT intervalelongation include: CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A,CYP3A5 and CYP3A7.

[0047] A further object of the invention is to provide a method fordetermining whether a subject has a predisposition for QT intervalelongation (e.g., acquired LQTS) when treated with one or morepharmaceutical agents comprising the step of screening a biologicalsample from the subject through a nucleic acid array, such as a DNAarray. The DNA array contains probes for two or more genetic mutationsor polymorphisms in at least two or more groups of genes wherein thegenes are selected from the group consisting of (1) LQT genes, (2)altered sensitivity genes (e.g., MiRP-1genes and its related genes), and(3) increased exposure genes (e.g., multidrug resistant genes andcytochrome P450 genes). The two or more genetic mutations orpolymorphisms are found in these genes as at least one or more geneticmutations or polymorphisms in each of the two or more groups of genes.The genes can be selected from those described above.

[0048] Another object of the invention is to provide a nucleic acidarray comprising nucleic acids which recognize and bind to mutations ofthe QT syndrome genes (e.g., LQT genes), the altered sensitivity genes(MirR-1 genes) and/or the increased exposure genes.

[0049] Another object of the invention is to provide a method ofscreening one or more pharmaceutical agents in vitro for its or theirability to induce prolonged cardiac repolarization of a cell comprisingthe steps of A) measuring I_(Kr) and I_(Ks) currents of the cell using avoltage clamp before superfusing the cell with a candidate agent oragents; B) superfusing and incubating the cell with the candidate agentor agents; C) measuring the I_(Kr) and I_(Ks) currents after superfusionand incubation of tie cell with the candidate agent or agents using avoltage clamp; and D) determining whether the I_(Kr) and I_(Ks) currentsare inhibited or abolished thereby indicating that the drug prolongsrepolarization.

[0050] It is another object of the invention to provide a method foridentifying genetic polymorphisms and mutations, which can cause QTinterval prolongation in a subject comprising the steps of inserting atleast two nucleic acids each encoding a polymorphism or mutation of atleast two of the following genes: a LQT gene, an altered sensitivitygene, and/or an increased drug exposure gene into a cell; B) measuringI_(Kr) and I_(Ks) currents of the cell before administering a drug knownto cause a change in I_(Kr) and/or I_(Ks); C) measuring I_(Kr) andI_(Ks) currents of the cell after superfusion of the cell with the drug;D) comparing the I_(Kr) and I_(Ks) values of the cell expressing thepolymorphisms and/or mutations to the I_(Kr) and I_(Ks) values of a cellexpressing a wild-type genes; and E) determining if the presence of thepolymorphisms and/or mutations leads to greater inhibition or blockageof I_(Kr) and I_(Ks) currents in the cell expressing said polymorphismor polymorphisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051]FIG. 1. Recordings of I_(Kr), I_(to) (A and B) and I_(K1) (C) inthe same cell before and after 5 minutes superfusion with 5 μmol/LE-4031. Panel A) I_(Kr) and I_(to) currents before and after superfusionwith E-4031. E-4031 abolished the I_(Kr) tail current and also reducedthe time-dependent I_(Kr) current without affecting the transientoutward current (I_(to) ) or the holding current; Panel B) E-4031sensitive currents obtained by digital subtraction of currents afterE-4031 exposure from currents before E-4031 exposure. Note the inwardrectification of the time-dependent I_(Kr) currents at very positivepotentials compared with the tail currents; Panel C) I_(K1) currentbefore and after superfusion with E-4031. E-4031 showed little effect onthe inward I_(K1) current recorded at −120 mV. The outward holdingcurrents that represent the amplitude of I_(K1) at −40 mV aresuperimposed.

[0052]FIG. 2. Recordings of I_(Kr), I_(to), (A and B) and I_(K1) (C) inthe same cell before and after 5 minutes superfusion with 10 μmol/Ltamoxifen. Panel A) I_(Kr) and I_(to) currents recorded before and aftersuperfusion with tamoxifen. Tamoxifen abolished the I_(Kr) tail currentand also reduced the time-dependent I_(Kr) current, without affectingthe transient outward current (I_(to)); Panel B) Tamoxifen-sensitivecurrents obtained by digital subtraction of currents before and aftertamoxifen superfusion. Note the inward rectification of thelime-dependent I_(Kr) currents at very positive potentials compared withthe tail currents and their similarity to the E-4031 sensitive currents;Panel C) I_(K1) current recorded before and after superfusion withtamoxifen. Tamoxifen showed no block of I_(K1) inward current. Theoutward holding currents, which represent the amplitude of I_(K1) at −40mV, were superimposed together.

[0053]FIG. 3. Time-dependent block of I_(Kr) by tamoxifen. I_(Kr)currents were recorded in the same cell before drug administration, 3, 5and 9 min. after superfusion with 1 μmol/L tamoxifen.

[0054]FIG. 4. Voltage- and concentration-dependent block of I_(Kr) bytamoxifen. I_(Kr) tail currents were measured 5-7 min. after superfusionwith tamoxifen. Panel A) Effect of 1 μmol/L I-V relationship; Panel B)Effect of 3.3 μmol/L tamoxifen on I-V (current voltage) relationship.Data are expressed as mean ±SD, n=4, ^(*)p<0.05.

[0055]FIG. 5. Comparison of I_(Kr) , block by tamoxifen and quinidine.Panel A) I_(Kr) currents recorded from the same cell before drugadministration, 5 minutes after superfusion with 10 μmol/L tamoxifen and5 minutes after washout. I_(Kr) tail currents were abolished bytamoxifen without recovery. Panel B) I_(Kr) currents recorded fromanother cell before drug administration. 5 minutes after superfusionwith 10 μmol/L quinidine and 3 minutes after washout demonstrate thatI_(Kr) tail currents were reduced but not abolished by quinidine withpartial recovery after 3 minutes washout. Panel C) inhibition of I_(Kr)by 3.3 μmol/L tamoxifen and 3.3 μmol/L quinidine. Data are expressed asmean ±SD, n=4, **p<0.01.

[0056]FIG. 6. Effect of tamoxifen on the action potential duration(APD). Action potentials were elicited by injecting 100 pA depolarizingcurrents of 2 ms, at a frequency of 0.45 HZ. Shown in the figure are twosuperimposed action potential tracings recorded in a single ventricularmyocyte before and 4 minutes after exposure to 3.3 μmol/L tamoxifen. Thetwo tracings are averaged tracings from 16 trials.

[0057]FIG. 7. Effect of t1tamoxifen on the L-type I_(Ca). I_(Ca) wasrecorded in the same cell before tamoxifen administration and 1, 2, 3, 4min. after superfusion of 10 μmol/L tamoxifen and after 2, 4, 8 and 16min. after washout. Note the marked inhibition of I_(Ca) and partialrecovery after washout.

[0058]FIG. 8. Baseline QTc intervals in abdomen during the three phasesof the menstrual cycle and in men. Bars indicate means and SEM, n=20males and 20 females.

[0059]FIG. 9. Change in QTc in males and females (menstrual phase).

[0060]FIG. 10. Role of delayed rectifier potassium currents oilspontaneously beating cardiomyocytes. Panel A. Effect of chromanol 293Bon the spontaneous beating rate in cultured neonatal rat cardiomyocytes.Panel B. Effect of E-4031 (10 μM) alone and in combination with 293 (μM)and isoproterenol (1 μM) on the spontaneous beating rate of culturedneonatal rat cardiomyocytes.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The invention involves a method for diagnosing a subject'spredisposition to an adverse drug response involving a prolonged QTinterval, resulting from an excessive accumulation of drugs due togenetic polymorphisms or mutations in at least two classes of genes,which can result in potentially fatal cardiac arrhythmic. The drugscover an array of pharmaceuticals including anti-arrhythmics,anti-psychotics, antidepressants, anti-anginals, antibiotics,anti-fungals, anti-virals, diuretics, migraine drugs, mental illnesstherapeutics, breast cancer therapeutics, anxiolytics, anti-nauseaagents, cardiac medication, opiate agonists, antihypertensives,antiinfectives, and anticonvulsants. The inventive method fordetermining the adverse drug reaction potential utilizes a DNA array(e.g., DNA chip), which can be used to assay a biological sample from apat example, a patient's DNA sample could be run through a DNA array todiagnose whether the patient has any genetic mutations or polymorphismsthat are associated with prolonging cardiac repolarization. Preferredgenes, which are associated directly or indirectly with prolongingrepolarization, include LQT genes, altered sensitivity genes (e.g.,MiRP1 genes or related genes), and increased exposure genes (cytochromeP450 genes and MDR genes).

[0062] 1. Definitions

[0063] By “bp” or “base pair” is meant the hydrogen bonded purine andpyrimidine pair in a double-stranded nucleic acid. Typically in DNA, thepairs are adenine (A) and thiamine (T), and guanine (G) and cytosine(C). In RNA, the pairs are adenine (A) and uracil (U), and guanine (G)and cytosine (C).

[0064] By “nucleotide” or “nt” is meant the nucleotide, typically adeoxyribonucleic acid, of the type adenine (A), thiamine (T), guanine(G), uracil (U), and cytosine (C) typically in the sense or codingorientation, but can also include antisense orientations of the gene orcoding sequence.

[0065] By “nucleic acid” or “nucleic acid molecule” is meant adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble- stranded form, and unless otherwise limited, would encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally occurring nucleotides.

[0066] By “aa” is meant amino acid.

[0067] By “gene” is meant a unit of inheritance that occupies a specificlocus on a chromosome (chr.), the existence of which can be confirmed bythe occurrence of different allelic forms. Preferred genes of thisapplication are those which impact cardiac repolarization, especiallythose which act to prolong cardiac repolarization, genes which determinethe elimination of drugs from a host, and genes which prolong the timenecessary to eliminate a drug from the host. This can include cytochromeP450 genes and ion channel genes.

[0068] By “mutation” is meant a one or more nucleotide change in the DNAor RNA sequence of an organism. For example, such mutation can be aframe-shift mutation, a nonsense mutation, or a missense mutation.

[0069] By “polymorphism” is meant the existing, in a population, of twoor more alleles of a gene, wherein the frequency of the rarer alleles isgreater than can be explained by recurrent mutation alone (typicallygreater than one percent). Said polypmorphisms can consist of one ormore nucleotide differences. The polymorphisms can be silent, whereinthey do not confer a change in the associated amino acid sequence.Alternatively, the polymorphism can cause an associated change in theamino acid sequence encoded by the gene.

[0070] By “altered sensitivity genes” is meant to include genes, whichwhen mutated, alter the expression of proteins which thereupon resultsin altered sensitivity to a drug or to drugs. Such genes can include forexample, the potassium ion channel gene, MiRP1 or related genes.

[0071] By “increased exposure gene” is meant to include genes which whenaberrantly expressed in a subject lead to increased exposure to a drugor drugs. Such genes can include cytochrome P450 genes, which whenmutated lead to decreased or aberrant expression of enzymes required forthe elimination of drugs or of a drug. Increased exposure genes alsoinclude multidrug resistance (MDR) genes. Mutations in MDR genes canlead to altered distribution (and thereby increased exposure) of a drugor drugs in tissues or in a tissue. MDR genes encode membrane drugtransporters and/or ion channel proteins.

[0072] By “ion channel gene” is meant to include multidrug resistancegenes (MDR genes) as well as ion pump genes such as the LQT family ofgenes, certain sodium (Na⁺) channel genes (see, e.g., Chen et al.,Nature 392: 293-6 (1998)) and certain potassium (K⁺) channel genes. Forexample, the potassium ion channel -gene, MiRP1, is one preferredexample of a potassium ion channel which may be linked to QT intervalprolongation; MiRP1 protein forms channels with HERG and its mutationsare associated with cardiac arrhythmia. Preferred MDR genes includeMDR-1 which encodes P-glycoprotein pump (P-gp).

[0073] By “prolonged QT interval,” “QT interval prolongation” or “QTinterval elongation” is meant the QT interval measured from QRS onset toT wave offset (QTo) and from QRS onset to T wave peak (QTm) adjusted toa heart rate of 60 beats per minute, which is QTc. By “QTc” is alsoreferred to as the Bazett corrected QT interval. See, e.g., Kligfield etal., J. Am. Coll. Cardiol. 28: 1547-55 (1996). Prolonged QT intervalscan be induced directly or indirectly by at least two genetic mutationsor polymorphism. These mutations or polymorphisms are located in two ormore groups of genes (e.g., at least one mutation or polymorphism pergene group), wherein the groups are (1) LQT genes, (2) alteredsensitivity genes (e.g., MiRP1 genes), or (3) increased exposure genes(e.g., MDR genes or cytochrome P450 genes).

[0074] By “Torsades de Pointes” or “TdP” is an uncommon variant ofventricular tachycardia (VT). The underlying etiology) and management ofTdP are, in general, quite different from the more common ventriculartachycardia. TdP is a polymorphous ventricular tachycardia in which themorphology of the QRS complexes vary from beat to beat. The ventricularrate can range from about 150/min to about 250/min. In most cases, thereis a constantly changing wave form, but there may not be regularity tothe axis changes. The definition also requires that the Q-T interval bemarkedly increased (usually to 600 msec or greater). Cases ofpolymorphic VT, which are not associated with a prolonged Q-T interval,are treated as generic VT. TdP usually occurs in bursts that are notsustained, thus, one usually has a rhythm strip showing the patient'sbase-line Q-T prolongation

[0075] By “predisposition” is meant a tendency for a subject to developTdP de Pointes or QT interval elongation. This tendency may be acquiredor hereditary. The preferred subject is a human subject. Thepredisposition is related to induction of TdP or QT interval elongationupon the administration of one or more pharmaceutical agents whichinduce TdP or QT interval elongation. These pharmaceutical agents can bethose listed herein or any later identified investigational drug whichinduces QT interval prolongation.

[0076] By “LQTS” or “long QT syndrome” is meant a genetic disease whichpredisposes individuals to ventricular arrhythmia that lead to syncopeand sudden death. Congenital or idiopathic LQTS is an inherited form ofthe disease and is genetically heterogeneous (Wei et al., Circulation92:1-275 (1995)) and includes the Jervell-Lange-Nielsen and theRomano-Ward-syndromes (Napolitano et al., Drugs 47: 51-65 (1994)).Acquired prolonged QT syndromes are largely iatrogenic, and may beinduced by certain drugs or associated with metabolic disturbances(e.g., hypokalemila., hypocalcemia or hypomagnesemia) (Napolitano etal., 1994).

[0077] By “subject,” “patient,” or “individual” is meant a mammal,especially a human.

[0078] By “nucleic acid array” is meant a substrate to which one ormore, preferably 50 or more, more preferably 100- 1,000 or more, andmore preferably 500 to 5,000 or more nucleic acids are attached. Also,contemplated are arrays with 5.000 to 500,000 nucleic acids attached.One example of such an array is a DNA chip array. For example, see U.S.Pat. Nos. 5,981,956 and 5.922,591. Other examples include Gene Logic'sFlow-thru ChipÔ Probe ArraysÔ (U.S. Pat. No. 5,994,068) or theFlowMetrix technology (e.g., microspheres) of Luminex. These arrays arecontemplated to contain nucleic acids for wild-type and mutated genesencoding ion channel genes and/or cytochrome P450 genes or isoformsthereof.

[0079] By “mutation,” “mutant” or “mutated” is meant to refer to agenetic change (e.g., frame-shift mutation, non-sense mutation, missensemutation, deletion, or insertion) in a gene (e.g. ion channel gene, P-gpor a cytochrome P450 gene) resulting in an altered gene expressionand/or altered protein function.

[0080] By “isoform” is meant different forms of a protein that may beproduced from different genes or from the same gene by alternative RNAsplicing.

[0081] By “binds substantially” is meant to complementary hybridizationbetween an oligonucleotide and a target sequence. By “hybridizing” ismeant the binding of two single stranded nucleic acids via complementarybase pairing.

[0082] The term “primer” refers to an oligonucleotide, whether naturalor synthetic capable of acting as a point of initiation of DNA synthesisunder conditions in which synthesis of a primer extension productcomplementary to a nucleic acid strand is induced, i.e., in the presenceof four different nucleotide triphosphates and an agent forpolymerization (i.e., DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. A primer is preferablya single-stranded oligodeoxyribo-nucleotide. The appropriate length of aprimer depends on the intended use of the primer, but typically rangesfrom 15 to 30 nucleotides. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with atemplate. The term “primer” may refer to more than one primer,particularly in the case where there is some ambiguity in theinformation regarding one or both ends of the target region to beamplified. For instance, if a region shows significant levels ofpolymorphism or mutation in a population, mixtures of primers can beprepared that will amplify alternate sequences. A primer can be labeled,if desired, by incorporating a label delectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (as commonly used in an ELISA), biotin, or haptens andproteins for which antisera or monoclonal antibodies are available. Alabel can also be used to “capture” the primer, so as to facilitate theimmobilization of either the primer or a primer extension product, suchas amplified DNA, on a solid support.

[0083] As used herein and unless described otherwise, “pharmaceuticalagent,” refers to an agent or drug which alone or in combination withone or more other pharmaceutical agents can induce in a patientprolonged cardiac repolarization. The specific pharmaceutical agentswhich may induce QT interval elongation are provided herein.

[0084] By “I_(K1)” is meant the major rapid repolarizing current in acell also known as rapid component of the delayed rectifier potassiumcurrent. By “I_(Ks) ” is meant the slower component of the delayedrectifier current. By “I_(K1) ” is meant inward rectifier current. BothI_(Kr) and I_(K1) are forms of potassium current densities. By “I_(to)”is meant the transient outward current of a cell as measured in avoltage clamp assay.

[0085] By “biological sample” or “sample” is meant a collection ofbiological material from a subject containing nucleated cells. Thisbiological material may be solid tissue, for example from a fresh orpreserved organ or tissue sample, biopsy or buccal swab; blood or bloodconstituents; bodily fluids such as amniotic fluid, peritoneal fluid, orinterstitial fluid, etc. The sample may contain compounds which are notnaturally intermixed with the biological material such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.

[0086] II. Pharmaceutical Agents

[0087] One embodiment of the invention is to identify a pharmaceuticalagent or combination of agents that induces QT interval elongation in asubject, especially a human. Agents or combinations of agents thatinduce elongation of the QT interval include, but are not limited to,those listed in Table 2, below. Several of these drugs have beenanalyzed and have been identified which prolong the QT interval in aconcentration-dependent manner (see, e.g., Drici et al., J. Clin.Psychopharmacol. 18: 477-81 (1998)). TABLE 2 Pharmaceutical Agents DrugBrand Name Drug Class QT^(†) TdP^(†) Amiodarone Cordarone ®Antiarrhythmic Yes Yes Amitriptyline Elavil ®, Endep ® AntidepressantYes Yes Amitriptylme Etrafon ® Antidepressant- HCl- AntipsychoticPerphenazine Amoxapine Asendin ® Antidepressant Yes AstemizoleHismanal ® Antihistamine Yes Yes Azelastine Astelin ® Antihistamine YesBepridil Vascor ® Antianginal Yes Yes Chlorpromazine Thorazine ® Mentalillness, Yes Yes nausea/vomiting Cisapride Propulsid ® Stimulates YesYes intestinal motility Clarithromycin Abbotic, Biaxin ®, Antibiotic YesBicrolid, Clacine, Clambiotic, Claribid, Clarith, Klacid, Klaricid,Klarin, Macladin, Naxy, Veclam Clemastine Tavist ® AntihistamineMaybe_(—) Clomipramine Anafranil ® Mental illness Yes DesipramineNorpramin ® Antidepressant Yes Diphenhydramine Benadryl ® AntihistamineMaybe_(—) Disopyramide^(‡) Norpace ® Antiarrhythmic Yes Yes DoxepinSinequan ®, Zonalon ® Aniidepressant Yes Yes Erythromycin^(‡)(Akne-Mycin ®, E.E.S. ®, Antibiotic and Yes Yes EryDerm ®, Erygel ®,intestinal Ery-Tab ®, Erythrocin ®, stimulant Erythromycin BaseFilmtab ®, Erythrostatin ®, E-mycin, EryPeds, PCE Felbamate Felbatrol ®Anticonvulsant Yes? Yes Flecainide Tambocor ® Antiarrhythmic Yes YesFluconazole Diflucan ® Antifungal Fludrocortisone Florinef ® Maintainblood Yes pressure/retain sodium Fluoxetine Prozac ® Antidepressant YesFluphenazine Prolixin ® Mental illness, Yes Yes Parkinson's DiseaseFluvoxamine Luvox ® Antidepressant Foscarnet Foscavir ® Antiviral YesFosphenytoin Cerebyx ® Hydanloin Yes Halofantrine^(‡) Antimalarial YesYes Haloperidol Haldol ® Mental illness, Yes Yes agitation Ibutilide^(‡)Corvert ® Antiarrhythmic Yes Yes Imipramine Tofranil ® AntidepressantYes Indapamide Lozol ® Diuretic Yes Maybe_(—) Isradipine Dynacirc ®Cardiac Drug Yes Itraconazole Sporanox ® Antifungal, AntibioticKetoconazole Nizoral ® Antifungal Levomethadyl Orlaam ® Opiate agonistYes Maprotiline Ludiomil ® Antidepressant Yes Yes Moexipril/HCTZUniretic ® Antihypertensive Yes Moricizine Ethmozine ® AntiarrhythmicYes Naratriptan Amerge ® Migraine therapy Yes Nicardipine Cardene ®Cardiac drug Yes Nortriptyline Pamelor ®, Aventyl ® Antidepressant YesOctreotide Sandostatin ® Unclassified Yes Pentamidin^(‡) Pentacarinat ®,Pentam ®, Antiinfective Yes Yes NebuPent ® Perphenazine Trilafon ®Mental illness Yes Yes Pimozide^(‡) Orap ® Tourette's Yes Yes syndrome,seizures Probucol^(‡) Lorelco ® Lowers cholesterol Yes Yes ProcainamideProcan ®, Procanbid ®, Antiarrhythmic Yes Yes Pronestyl ®Prochlorperazine Compazine ® Nausea Maybe_(—) Protriptyline Vivactil ®Antidepressant Yes Quetiapine Seroquel ® Antipsychotic Yes Quinidine^(‡)Cardioquin ®, Antiarrhymic Yes Yes Duraquin ®, Quinidex ®, Quinaglute ®Risperidone Risperdal ® Mental illness Yes Yes Salmeterol Serevent ®Sympathomimetic- Yes Adrenergic Sotalol^(‡) Betapace ® AntiarrhythmicYes Yes Sparfloxacin Zagam ® Antibiotic Yes Yes (pneumonia andbronchitis) Sumatriptan Immitrex ® Migraine Yes treatment TamoxifenNolvadex ® Breast cancer Yes therapeutic Terfenadine^(‡) Seldane ®Antihistamine Yes Yes Thioridazine Mellaril ® Mental illness Yes YesThiothixene Navane ® Mental illness Yes Yes Tizanidine Zanaflex ® Musclerelaxant Yes Tocainide Tonocard ® Antiarrhythmic Yes TrifluoperazineStelazine ® Mental illness Yes Yes Trimethoprim Bactrim ®, Septra ®,Aniibiotic Maybe_(—) Sulfamethoxazole Trimeth-Sulfa ® VenlafaxineEffexor ® Antidepressant Yes Zolmitriptan Zomig ® Migraine Yes Treatment

[0088] In addition to adverse reactions in a subject to a singlepharmaceutical agent, such as those listed above, certain of theseagents may induce adverse reactions in specific subjects only whencombined with one or more other agents. This is due to two or moregenetic polymorphisms located in two or more classes of genes. Themutations or polymorphisms would appear in one or more of the genes intwo or more of the classes of genes. The classes of genes include (1)LQT genes, (2) altered sensitivity genes and (3) increased exposuregenes. For example, the mutations could occur in one LQT gene and onecytochrome P450 gene or in MiRP1 and LQT3. The polymorphisms ormutations generally cause aberrant enzyme activity resulting in anadverse drug reaction either due to altered sensitivity to the drugs orincreased exposure to the drugs. Drugs which likely are involved inadverse drug-drug interactions due to polymorphisms in part in the P450genes are listed in Table 3 below. TABLE 3 Drug Interactions Induced inpart by Cytochrome P450 Genes Drug Brand Name Interactions^(†) QT TdPAmiodarone Cordarone ® 1A2 Inhibitor Yes Yes 2C9 Inhibitor 2D6 Inhibitor3A Inhibitor Amitriptyline Elavil ®, Endep ® 1A2 Substrate Yes Yes 2C19Substrate 2C9 Substrate 2D6 Substrate Astemizole Hismanal ® 3A SubstrateYes Yes Cisapride Propulsid ® 3A Substrate Yes Yes ClarithromycinAbbotic, Biaxin ®, 3A Substrate Yes Bicrolid, Clacine, 3A InhibitorClambiotic, Claribid, Clarith, Klacid, Klaricid, Klarin, Macladin, Naxy,Veclam Clemastine Tavist ® Interactions Maybe_(—) ClomipramineAnafranil ® 1A2 Substrate Yes 2C19 Substrate 2D6 Substrate 2D6 InhibitorDesipramine Norpramin ® 2D6 Substrate Yes Erythromycin^(‡)(Akne-Mycin ®, E.E.S. ®, 3A Substrate Yes Yes EryDerm ®, Erygel ®, 3AInhibitor Ery-Tab ®, Erythrocin ®, Erythromycin Base Filmtab ®,Erythrostatin ®, E-mycin, EryPeds, PCE Felbamate Felbatrol ® 2C19Inhibitor Yes Yes Flecainide Tambocor ® 2D6 Substrate Yes YesFluconazole Diflucan ® 2C9 Inhibitor 3A Inbibilor Fluoxetine Prozac ®2C9 Substrate Yes 2D6 Substrate 2C19 Inhibitor 2D6 Inhibitor 3AInhibitor Fluphenazine Prolixin ® Interactions Yes Yes FluvoxamineLuvox ® 1A2 Substrate 2D6 Substrate 1A2 Inhibitor 2C19 Inhibitor 2C9Inhibitor 3A Inhibitor Halofantrine^(‡) 2D6 Inhibitor Yes YesHaloperidol Haldol ® 1A2 Substrate Yes Yes 2D6 Substrate 3A Substrate2D6 Inhibitor Imipramine Tofranil ® 1A2 Substrate Yes ItraconazoleSporanox ® 3A Inhibitor Ketoconazole Nizoral ® 2C19 InhibitorProtriptyline Vivactil ® Interactions Yes Quinidine^(‡) Cardioquin ®, 3ASubstrate Yes Yes Duraquin ®, Quinidex ®, 2D6 Inhibitor Quinaglute ®Risperidone Risperdal ® 2D6 Substrate Yes Yes Tamoxifen Nolvadex ® 2C9Substrate Yes Terfenadine^(‡) Seldane ® 3A Substrate Yes YesThioridazine Mellaril ® 2D6 Substrate Yes Yes Trimethoprim Bactrim ®,Septra ®, Potential 2C9 Maybe_(—) Sulfamethoxazole Trimeth-Sulfa ®Inhibitor Venlafaxine Effexor ® 2D6 Substrate Yes

[0089] III. Kits and Methods of Diagnosing Patients with aPredisposition for QT Interval Elongation

[0090] Although more than 120 mutations have been described in patientswith LQTS, not all of these mutations cause QT internal prolongation ina subject after the administration of a specific pharmaceutical agent oragents. Some gene mutations responsible for QT interval prolongation ina subject include, but are not limited to, those listed in Table 7below. TABLE 7 aa nt Mutation Gene Position Position Type *Reference¹CYP2D6* ¹⁹³⁴G ® Splice site (53)Oscarson et al., Mol. 4 A defectPharmacol. 52: 1034-40 (1997)); Topic et al., Clin. Chem. Lab. Med. 36:655-8 (1998) CYP2D6* C188CIT Someya et al., Psychiatry 10 in exon 1Clin. Neurosci. 53: 593-7 (1999) CYP2D6* ¹¹¹¹C ®T point Masimirembwa etal., Br. 17/*17 ²⁹³⁸C ®T mutations Clin. Pharmacol. 42: ⁴²⁶⁸G ®C 713-9(1996) HERG 593 1778 point Benson et al., Circulation mutation 93:1791-5 (1996) KCNE1  76  226 point Schulze-Bahr et al., Nature mutationGenet. 17: 267-8 (1997) KCNQ1  73  217 point Donger et al., Circulationmutation 96: 2778-81 (1997) KCNQ1  95  284 point Wang et al., NatureGenet. mutation 12: 17-23 (1996) KCNQ1 159  475 point Wang. et al.,(1996) mutation KCNQ1 174  521 point Donger et al., (1997) mutationKCNQ1 210  629 point Neyroud et al., Eur. J. mutation Hum. Genet. 6:129-33 (1998) KCNQ1 219  655 point Russell et al., Hum. Molec. mutationGenet. 5: 1319-24 (1996) KCNQ1 220  659 point Donger et al., (1997)mutation KCNQ1 246  737 point Wang. et al., (1996) mutation KCNQ1 249 746 point Donger et al., (1997) mutation KCNQ1 460 1378 point Donger etal., (1997) mutation KCNQ1 415 1244- deletion- Neyroud et al., (1998)1250 insertion SCN5A  1505- 4513- 9 bp Wang. Hum. Molec. Genet. 15074521 deletion 4: 1603-7 (1995)

[0091] Nucleic acids which recognize these mutations can be placed in anarray on a substrate, such as on a chip (e.g., DNA chip or microchips).These arrays also can be placed on other substrates, such as microtiterplates, beads or microspheres. Methods of linking nucleic acids tosuitable substrates and the substrates themselves are described, forexample, in U.S. Pat. Nos. 5,981,956; 5,922.591; 5,994,068 (Gene Logic'sFlow-thru ChipÔ Probe ArraysÔ). 5,858,659, 5,753,439: 5,837,860 and theFlowMetrix technology (e.g., microspheres) of Luminex (U.S. Pat. Nos.5,981,180 and 5,736,330).

[0092] The nucleic acids that recognize the polymorphisms of the LQT andcytochrome P450 genes preferably can be linked to a single substrate.Alternatively, in the case of microspheres, the substrate may onlycomprise a single or a few (e.g., 2, 3, 4, 5, or 10) nucleic acids andmay be mixed with microspheres comprising different nucleic acids.

[0093] There are two preferred methods to make a nucleic acid array. Oneis to synthesize the specific oligonucleotide sequences directly ontothe solid-phase in the desired pattern (Southern et al., Nucl. AcidsRes. 22: 1368-73 (1994): Maskos et al., Nucl. Acids Res. 20: 1679-84(1992); Pease et al., Proc. Natl. Acad. Sci. 91: 5022-6 (1994): and U.S.Pat. No. 5,837.860) and the other is to presynthesize theoligonucleotides in an automated DNA synthesizer and then attach theoligonucleotides onto the solid-phase support at specific locations(Lamture et al., Nucl. Acids Res. 22: 2121-5 (1994) and Smith et al.,Nucl. Acids Res. 22: 5456-64 (1994)). In the first method, theefficiency of the coupling step of each base affects the quality andintegrity of the nucleic acid molecule array.

[0094] A second, more preferred method for nucleic acid array synthesisutilizes an automated DNA synthesizer for DNA synthesis. The controlledchemistry of an automated DNA synthesizer allows for the synthesis oflonger, higher quality DNA molecules than is possible with the firstmethod. Also, the nucleic acid molecules synthesized can be purifiedprior to the coupling step. The nucleic acids can be attached to thesubstrate as described in U.S. Pat. No. 5,837.860.

[0095] A. Hybridization Detection of PCR Products

[0096] Thus, for example, covalently immobilized nucleic acid moleculesmay be used to detect specific PCR products by hybridization where thecapture probe is immobilized on the solid phase or substrate (Ranki etal., Gene 21: 77-85 (1983); Keller et al., J. Clin. Microbiol. 29:638-41 (1991); Urdea et al., Gene 61: 253-64 (1987)). A preferred methodwould be to prepare a single-stranded PCR product before hybridization.A patient sample that is suspected to contain the target molecule, or anamplification product thereof, would then be exposed to thesolid-surface and permitted to hybridize to the bound oligonucleotide.

[0097] The methods of the present invention do not require that thetarget nucleic acid contain only one of its natural two strands. Thus,the methods of the present invention may be practiced on eitherdouble-stranded DNA (dsDNA), or on single-stranded DNA (ssDNA) obtainedby, for example, alkali treatment of native DNA. The presence of theunused (non-template) strand does not affect the reaction.

[0098] Where desired, however, any of a variety of methods can be usedto eliminate one of the two natural stands of the target DNA moleculefrom the reaction. Single-stranded DNA molecules may be produced usingthe ssDNA bacteriophage, M13 (Messing et al., Meth. Enzymol. 01: 20-78(1983); see also, Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)).

[0099] Several alternative methods can be used to generatesingle-stranded DNA molecules. For example, Gyllensten et al., Proc.Natl. Acad. Sci. U.S.A. 85: 7652-6 (1988) and Mihovilovic et al.,BioTechiques 7: 14-6 (1989) describe a method, termed “asymmetric PCR,”in which the standard “PCR” method is conducted using primers that arepresent in different molar concentrations.

[0100] Other methods have also exploited the nuclease resistantproperties of phosphorothioate derivatives in order to generatesingle-stranded DNA molecules (U.S. Pat. No. 4,521,509); Sayers et al.,Nucl. Acids Res. 16: 791-802 (1988); Eckstein et al. Biochemistry 15:1685-91 (1976); and Ott et al., Biochemistry 26: 8237-41 (1987): andSAMBROOK et al.,1989).

[0101] C. Screening Polymorphisms

[0102] Screening polymorphisms in samples of genomic material accordingto the methods of the present invention, is generally carried out usingarrays of oligonucleotide probes. These arrays nay generally be “tiled”for a large number of specific polymorphisms. By “tiling” is generallymeant the synthesis of a defined set of oligonucleotide probes which ismade up of a sequence complementary to the target sequence of interest,as well as preselected variations of that sequence, e.g., substitutionof one or more given positions with one or more members of the basic setof monomers. i.e. nucleotides. Tiling strategies are discussed in detailin Published PCT Application No. WO 95/11995, incorporated herein byreference in its entirety for all purposes. By “target sequence” ismeant a sequence which has been identified as containing a polymorphismor mutation (e.g., a single-base polymorphism also referred to as a“biallelic base”). It will be understood that the term “target sequence”is intended to encompass the various forms present in a particularsample of genomic material, i.e., both alleles in a diploid genome.

[0103] In a particular aspect, arrays are tiled for a number ofspecific, identified polymorphic marker sequences. In particular, thearray is tiled to include a number of detection blocks, each detectionblock being specific for a specific polymorphic marker or set ofpolymorphic markers. For example, a detection block may be tiled toinclude a number of probes which span the sequence segment that includesa specific polymorphism. To ensure probes that are complementary to eachvariant, the probes are synthesized in pairs differing, for example, atthe biallelic base.

[0104] In addition to the probes differing at the biallelic bases,monosubstituted probes can be generally tiled within the detectionblock. These monosubstituted probes have bases al and up to a certainnumber of bases in either direction from the polymorphisms, substitutedwith the remaining nucleotides (selected from A, T, G, C or U).Typically, the probes in a tiled detection block will includesubstitutions of the sequence positions up to and including those thatare 5 bases away from the base that corresponds to the polymorphism.Preferably, bases up to and including those in positions 2 bases fromthe polymorphism will he substituted. The monosubstituted probes provideinternal controls for the tiled array, to distinguish actualhybridization from artifactual cross-hybridization.

[0105] A variety of tiling configurations may also be employed to ensureoptimal discrimination of perfectly hybridizing probes. For example, adetection block may be tiled to provide probes having optimalhybridization intensities with minimal cross-hybridization. For example,where a sequence downstream from a polymorphic base is G-C rich, itcould potentially give rise to a higher level of cross-hybridization or“noise,” when analyzed. Accordingly, one can tile the detection block totake advantage of more of the upstream sequence.

[0106] Optimal tiling configurations may be determined for anyparticular polymorphism by comparative analysis. For example, triplet orlarger detection blocks may be readily employed to select such optimaltiling strategies.

[0107] Additionally, arrays will generally be tiled to provide for easeof reading and analysis. For example, the probes tiled within adetection block will generally be arranged so that reading across adetection block the probes are tiled in succession, i.e., progressingalong the target sequence one or more nucleotides at a time.

[0108] Once an array is appropriately tiled for a given polymorphism orset of polymorphisms (e.g., LQT and cytochrome P450 genes), the targetnucleic acid is hybridized with the array and scanned. Hybridization andscanning are generally carried out by methods described in, e.g., PCTApplication Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No.5,424,186. In brief, a target nucleic acid sequence, which includes oneor more previously identified polymorphic markers, is amplified by wellknown amplification techniques, e.g., polymerase chain reaction (PCR).Typically, this involves the use of primer sequences that arecomplementary to the two strands of the target sequence both upstreamand downstream from the polymorphism. Asymmetric PCR techniques may alsobe used. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the position on the array to which the target sequencehybridizes. The hybridization data obtained from the scan is typicallyin the form of fluorescence intensities as a function of location on thearray.

[0109] Although primarily described in terms of a single detectionblock, e.g., for detection of a single polymorphism, in the preferredaspects, the arrays of the invention will include multiple detectionblocks, and thus be capable of analyzing multiple, specificpolymorphisms. For example, preferred arrays will generally include fromabout 50 to about 4,000 different detection blocks with particularlypreferred arrays including from 10 to 3,000 different detection blocks.

[0110] In alternate arrangements, it will generally be understood thatdetection blocks may be grouped within a single array or in multiple,separate arrays so that varying, optimal conditions may be used duringthe hybridization of the target to the array. For example, it may oftenbe desirable to provide for the detection of those polymorphisms thatfall within G-C rich stretches of a genomic sequence, separately fromthose falling in A-T rich segments. This allows for the separateoptimization of hybridization conditions for each situation.

[0111] Additional methods of detecting gene mutations (e.g.,polymorphisms) includes the methods described in International PCTapplications WO 99/42622; WO 99/29901; WO 98/49341; WO 97/27317; and W7097/22720.

[0112] D. Calling

[0113] After hybridization and scanning, the hybridization data from thescanned array is then analyzed to identify which variant or variants ofthe polymorphic marker are present in the sample, or target sequence, asdetermined from the probes to which the target hybridized, e.g., one ofthe two homozygote forms or the heterozygote form This determination istermed “calling” the genotype. Calling the genotype is typically amatter of comparing the hybridization data for each potential variant,and based upon that comparison, identifying the actual variant (forhomozygotes) or variants (for heterozygote) that are present. In oneaspect, this comparison involves taking the ratio of hybridizationintensities (corrected for average background levels) for the expectedperfectly hybridizing probes for a first variant versus that of thesecond variant. Where the marker is homologous for the first variant,this ratio will be a large number, theoretically approaching an infinitevalue. Where homozygous for the second variant, the ratio will be a verylow number i.e., theoretically approaching zero. Where the marker isheterozygous, the ratio will be approximately 1. These numbers are, asdescribed, theoretical. Typically, the first ratio will be well inexcess of 1, i.e., 2, 4, 5 or greater. Similarly, the second ratio willtypically be substantially less than 1, i.e., 0.5, 0.2, 0.1 or less. Theratio for heterozygotes will typically be approximately equal to 1,i.e., from 0.7 to 1.5. These ratios can vary based upon the specificsequence surrounding the polymorphism, and can also be adjusted basedupon a standard hybridization with a control sample containing thevariants of the polymorphism .

[0114] The quality of a given call for a particular genotype may also bechecked. For example, the maximum perfect match intensity can be dividedby a measure of the background noise (which may be represented by thestandard deviation of the mismatched intensities). Where the ratioexceeds some preselected cut-off point, the call is determined to begood. For example, where the maximum intensity of the expected perfectmatches exceeds twice the noise level, it might be termed a good call.Further description of software used for genetic calling can be used asdescribed in U.S. Pat. No. 5.858,659.

[0115] E. Method of Identifying New Polymorphisms

[0116] Another aspect of the invention is to identify polymorphisms ormutations which are associated with or indirectly involved with QTinterval prolongation. These polymorphisms or mutations are located inat least two classes of genes (e.g., LQT genes, altered sensitivitygenes or increased exposure genes). Moreover, the mutations orpolymorphisms can be those which have been previously identified but notlinked with QT interval elongation. Alternatively, once new mutations orpolymorphisms are identified, these also can be assessed using theassays described herein to determine whether the “new mutation” and/or“polymorphism” can cause QT interval elongation. As new polymorphismsand mutations are identified, the nucleic acids which recognize thesepolymorphisms can be added to the nucleic acid array for screeningsubjects. One method of identifying such “new” polymorphisms is toobtain biological samples from subjects who have experienced acquiredLQTS due to administration of a drug or drugs and to sequence the LQTgenes or P450 genes to isolate the polymorphism which was responsiblefor the adverse drug reaction.

[0117] Alternatively, for known mutations and polymorphisms in each ofthese gene classes, which previously has not been associated withadverse drug reactions, the drugs can be assessed for their ability toelongate the QT interval as discussed.

[0118] IV. Method of Identifying Agents Which Induce ProlongedRepolarization

[0119] Methods of identifying agents which prolong QT intervals can bepreformed as described in the examples provided below. Alternatively,agents can be assayed using the Langeudorff technique in, for example.,isolated perfused rabbit hearts, or the whole-cell patch-clamp techniquein ventricular myocytes to examine the TdP difference. Liu et al., J.Cardiovasc. Pharmacol. 34: 287-94 (1999) and Ebert et al., J. WomensHealth 7: 547-57 (1998) used these techniques to examine the genderdifference of Torsades de Pointes (TdP) between men and women. Drici etal., J Cardiovasc. Pharmacol., 34: 82-8 (1999) used isolatedLangendorff-perfused rabbit hearts to lest the effect of the agent,Tegaserod (HTF 91 9), on cardiac repolarization. Perfused (Langendorff)feline hearts can also be used, as described in Wang et al., J.Cardiovasc. Pharmacol. 32: 123-8 (1998), in which the authors used themodel system to assess QT prolongation in response to antihistamineadministration. The whole-cell patch-clamp technique was utilized tostudy the effect of tamoxifen on the delayed rectifier (I_(Kr)), theinward rectifier (I_(K1)) the transient outward current (I_(to)) and theinward L-type calcium current (I_(Ca)) in rabbit ventricular myocytes(Liu et al. J. Pharmacol. Exp. Ther. 287: 877-83 (1998)).

[0120] In addition to the above in vitro methods, determination of theeffect of a particular compound can also be performed by using anelectrocardiogram (ECG) to study the pharmokinietics of a drug's effecton QTC prolongation (see, e.g., Sale et al., Clin. Pharmacol. Ther. 56:295-301 (1994)).

[0121] Methods of measuring potassium (K⁺) currents in ATl cells and inoocytes are known in the art and can performed using the methods, forexample, described by Yang et al., Circulation91: 1799-1806 (1995); andDriga et al., Biophysics J. 74: A210 (1998). Basically, the cells aretransfected with a nucleic acid with the putative mutation believed tocause QT prolongation when exposed to an agent known to cause QTelongation. The I_(Kr) and I_(Ks) and even the I_(to) responses aremeasured and compared to a cell expressing the normal wild type gene.This can also be done when testing Na⁺current changes.

[0122] The effect of the genetic mutations can also be assessed usingvoltage sensitive dye methods. Assaying changes to cell voltage usingdyes are known in the art. See, for example, Morley et al., J.Cardiovasc. Electrophysiol. 10: 1361-75 (1999) and Dillon et al.,Science 214: 453-6 (1981).

[0123] The following examples are offered to illustrate embodiments ofthe invention, and should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Method and Kit for Determining a Subject'sRedisposition to Adverse Reactions to Tamoxifen.

[0124] The metabolic pathways of tamoxifen are complex and have beenextensively studied. Tamoxifen metabolism involves multiple pathways,and the primary and secondary metabolites have variable pharmacologicalactivities, with certain of the metabolites causing considerableinter-individual variability. In vitro and in vivo studies in humanshave shown that the main routes of tamoxifen metabolism includeN-demetlhylation, N-oxidation and 4-hydroxylationi (Buckley et al.,Drugs 37: 451-90 (1989); and Lim et al., Carcinogenic 15: 589-93(1994)). Tamoxifen N-demetlhylation, which appears quantitatively themost important pathway, is primarily catalyzed by CYP3A (Jacolot et al.,Biochem. Pharmacol. 41: 1911-9 (1991); (Mani et al., Drug Metab. Dispos.21: 645-56 (1993)). However CYP2D6 also appears to be a major enzymethat catalyzes tamoxifen 4-hydroxylation (Crewe et al., Biochem.Pharmacol. 53: 171-8 (1997)).

[0125] Methods for DNA extraction. Blood is drawn from a subject, e.g.,human, into sodium heparin Vacutainers (Becton Dickinson; FranklinLakes, N.J.) and then is transferred to Corning 5.0 ml cryogenic vials(Corning; Cambridge, Mass.). The blood is frozen in a non-frost free−20° C. freezer until use.

[0126] Reagents, which are used to extract the DNA from whole blood, arethe QIAGEN Blood Midi Kit(®) unless otherwise noted. 200 μl of QIAGENprotease is added to a 15 ml centrifuge tube. The blood is thawed and2.0 ml added to the tube followed by 2.0 ml of Buffer AL. The tube iscapped and contents mixed with a vortexer for 15 seconds. Aftervortexing, 2 ml of 95% ethanol is added, and the contents mixed byinversion. The contents of the tube is then added to a QIAGEN Midi Spincolumn that is placed in a collection tube. The column and collectiontube are centrifuged at 6.000×g at 4° C. for two minutes. Then thecollection tube is discarded and replaced with a new collection tube.Two ml of Buffer AW is added to the spin column, and the column iscentrifuged again at 6.000×g at 4° C. for two minutes. The column isrinsed again by discarding the collection tube, replacing it with a newcollection tube, adding 2.0 ml of Buffer AW to the spin column, andcentrifuging at 6.000×g at 4° C. for two minutes. The used collectiontube is discarded and replaced with a new collection tube. The DNA iseluted from the column by adding 1 ml of elution buffer to the spincolumn, incubating at room temperature for one minutes, and thencentrifuging at 6.000×2 at 4° C. for two minutes. The DNA is transferredto a Sarstedt 2.0 ml screw-top tube (#72730006, Sarstedt; Newton, N.C.)and frozen at −20° C.

[0127] The DNA concentration is measured using the 260/280 method in aspectrophotometer, and the DNA concentration adjusted to 60 ng/μl withwater.

[0128] DNA from buccal swabs is obtained as follows. Buccal cells areobtained by gently rubbing a sterile cotton swab within the subject'smouth. The swab is placed in a Falcon 2063 tube, and 1.5 mils of 1X PBSis added, mixed, and centrifuged at 3K for 5 minutes to pellet thecells. The supernatant is removed, and this process is repealed withanother 1 ml of 1X PBS. The cell pellet is then suspended in 47 μl ofPCR lysis buffer (Promega PCR Buffer B and 10 mg/ml Proteinase K).Samples are incubated for 30 minutes at 60° C. and the reaction stoppedby boiling the sample for 10 minutes. The sample is then centrifuged at3K for 5 minutes and stored at −20° C. until use.

[0129] CYP2D6 and CYP2C19 Genotyping Using the Affymetrix P450GeneChip®. Reagents used in this protocol use the Affymetrix P450GeneChip Kit® unless otherwise noted. The reaction mass mix is preparedin a template free area by combining the following: TABLE 4 PCRAmplification per reaction Final concentration H₂O 23 μl  AffymetrixCYP450 primer mix 4 μl 200 μM 20% DMSO 5 μl 2.0% 2.0 mM dNTP mix 5 μl200 μM AmpliTaq Gold ® 5 μl 1 X PCR buffer 25 mM MgCl₂ 5 μl 2.5 mM 5U/μl AmpliTaq Gold ® 1 μl 5 U Taq polymerase

[0130] TABLE 5 Primers CYP 2D6 Upstream Primer Downstream Primer Exon5′-CAGAGGAGCCCATTTGGTA 5′-GGTCCCACGGAAATCTGTC 1/2 GTG AGGCAGGT-3′TCTGT-3′ Exon 5′-CACGCGCACGTGCCCGTCC 5′-CTCTCGCTCCGCACCTCGC 3/4 CA-3′GCAGA-3′ Exon 5′-GGACTCTGTACCTCCTATC 5′-CCTCGGCCCCTGCACTGTT 5/6 CACGTCA-3′ TCCCA GA-3′ Exon 5′-GGCGACCAGAGATGGGTGA 5′-GCGCCAGGCCTACCTTAGG7/7 CCA GGCTC-3′ GATG CGGGA-3′ Exon 5′-GGGAGACAAACCAGGACCT5′-CATCTGCTCAGCCTCAACG 8/9 GC CAGA-3′ TACC CCTGTCT-3′

[0131] The required number of Micro-Amp® 8-strip Reaction Tubes areplaced in a 96-well tube/tray retainer and the assembly is placed in aMicroAmp® base (all Perkin-Elmer; Foster City. Calif.). forty-five μl ofthe mass mix is aliquoted into the 0.2 ml MicroAmp tubes and the tubesare capped. The tubes are then transferred to a medium template area toadd the DNA.

[0132] Five μl of DNA (60 ng/μl) is added to each sample tube; 5 μl ofAffymetrix CYP450 Reference DNA is added to the positive control tube;and 5 μl of water are added to negative control tube. The tubes are thencapped, and the rack centrifuged in a table top centrifuge at 2,000 rpmfor 1 minute.

[0133] The samples are then placed in a Perkin-Elmer GeneAmp® PCR System9600 thermocycler programmed for: 95° C. for 5 minutes, then 15 cyclesof 95° C./40 seconds, 65° C./50 seconds, 72° C./50 seconds followed by30 cycles of 95° C./30 seconds, 65° C./50 seconds, 72° C./50 secondsplus one second per cycle then a final extension of 72° C. for 7minutes.

[0134] A 10 μl aliquot of each product is loaded (with loading buffer)onto a 2% agarose. The gel is run until the bromophenol blue band is 2/3of the way down the gel, and photographed on a UV transilluminator.Correctly amplified products appear as bands of 159, 171, 250, 444, 762,878 and 1,125 base pairs. All bands must be present to obtain bothCYP2D6 and CYP2C19 calls. Failure of the larger two bands to amplifyprecludes any CYP2D6 call. If all bands are present then the next stepis that of fragmentation.

[0135] Fragmentation. A clean reaction tube rack is placed on ice withone tube for each sample that is to be fragmented. All fragmentationmass mix reagents are kept on ice, and the mass mix prepared bycombining the reagents as directed in Table 6. TABLE 6 25 μl mass mix250 μl mass mix Fragmentation reagent 1 μl 2 μl 20 mM EDTA 1 μl 2 μl 1U/μl alkaline 12.5 μl   25 μl   phosphatase water 110.5 μl    221 μl 

[0136] 125 μl mass mix for 1-25 reactions, 250 μl mass mix for 25-50reactions. Five μl of the fragmentation mass mix is added to each tube(on ice), and 10 μl of PCR product is added to the tubes. The tubes arecapped and either the rack tapped against a bench top briefly or quicklycentrifuged at 4° C. to bring the components together. The rack is thenplaced in a Perkin-Elmer GeneAmp® PCR System 9600 thermocycler and the25° C./20 minutes, 95° C./10 minutes, 4° C. hold fragmentation programis run.

[0137] Labeling. The labeling master mix is prepared by combining thereagents in a microcentrifuge tube as follows: TABLE 7 per reactionFinal Terminal Transferase Buffer   4 μl 1 X Fluorescein N6-ddATP 0.5 μl25 μM 20 U/μl Terminal Transferase 0.5 μl 10 U

[0138] Five μl of the labeling master mix is added to each tube offragmented product, and the rack placed in a Perkin-Elmer GeneAmp® PCRSystem 9600 thermocycler and the 37° C./35 minutes, 95° C./5 minutes, 4°C. hold labeling program is run.

[0139] Hybridization Preparation. The following solutions are preparedaccording to the direction in the Affymetrix® GeneChip® CYP450instruction booklet:

[0140] Hybridization Concentrate:

[0141] 5.5×SSPE, 0.055% Triton® X-100, and 1.1 mM CTAB

[0142] (hexadecyltrimethylammonium bromide, Sigma H-6269; St. Louis,Mo.)

[0143] Hybridization Master Mix:

[0144] Combine 45.5 ml hybridization concentrate, 1 ml molecular biologygrade water, 1 ml 50X Denhardt's Solution, and 500 μl Affymetrix®Control Oligonucleotide F1. Place 480 μl aliquots in 1.5 ml EppendorfSafe-Lock microcentrifuge tubes and freeze at −20° C. (the combinationof Denhardt's Solution and CTAB may cause background. If so, substitutemolecular biology grade water for the Denhardt's Solution).

[0145] Hybridization. Twenty μl of fragmented and labeled PCR product isadded to a tube of Hybridization Master Mix and labeled with the sample.The tubes are placed in boiling water for 10 minutes and thenimmediately transferred to ice. The tubes are kept on ice at least 10minutes. While the tubes are on ice, the Affymetrix® Fluidics Station isprimed with water. Wash Buffer A (3×SSPE, 0.005% Triton X-100 and 1 mMCTAB) and Wash Buffer B (6×SSPE).

[0146] A CYP450 Probe array is placed in the Fluidics Station module,and one of the Hybridization Master Mix/DNA tubes placed in the lowercompartment. The CYP450 Hybridization protocol is run on the FluidicsStation.

[0147] Scanning and Analysis. The hybridized chip is transferred fromthe Fluidics Station to the Scanner. A scan protocol is then run on thechip as directed by Affymetrix®. After the scan is completed, theAnalysis program is run and the report prepared. The results are enteredinto an appropriate database.

[0148] Conventional CYP2D6^(*)4 Assay, Amplification. Five μl of DNA (60ng/μl) is added to 20 μl of PCR reaction mix containing 1×PCR Buffer B(Promega; Madison, Wis.) [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.0%Triton X-100]; 25 pmol of each primer, 200 μM of each dNTP, 1.5 mM MgCl₂and 0.75 U of Taq polymerase (Promega; Madison, Wis.) In 500 μl PCRreaction tubes. Twenty five μl of mineral oil is added to the top ofeach reaction tube. The primers used are those of Heim et al, Metho.Enzymol. 206:173-83 (1991): 5′-TGC CGC CTT CGC CAA CCA CT-3′upstream and5′-GTG CGG AGC GAG AGA CCG AGG-3′ downstream. The tubes are brieflycentrifuged in a microcentrifuge and placed in a Perkin Elmer® model 480thermocycler. The amplification program used is 2 minutes at 94° C.denaturation; 35 cycles of 1 minute at 94° C.; 1 minute at 63° C.; 1minute at 72° C.; with a final extension of 4 minutes at 72° C.

[0149] Restriction Enzyme Digestion. A mass mix containing 2 μl of BstNI(NEB; Beverly, Mass.), 3 μl of NEB Buffer 2, and 0.3 μl of 100×BSA perreaction is prepared and 5.3 μl added to each reaction tube. The tubesare briefly centrifuged in a microcentrifuge to bring the restrictionenzyme mix below the mineral oil and incubated at 60° C. for at leastfour hours.

[0150] Gel Electrophoresis. The samples are electrophoresed on a 2.0%agarose gel. Wild-type allele produces 110 and 182 base pair fragments.The ^(*)4 mutant produces a 292 base pair fragment and a heterozygoteshows 110, 182 and 292 base pair bands on the gel.

[0151] Conventional CYP2D6^(*)10 Assay.

[0152] Amplification. The assay that is used for CYP2D6^(*)10 is a twoamplification allele specific oligonucleotide method.

[0153] Amplification 1. Five μl of DNA (60 ng/μl) is added to 45 μl PCRreaction mix containing 1×PCR buffer B (Promega; Madison, Wis.) [50 mMKCl, 10 mM Tris-HCl (pH 9.0), 1.0% Triton X-100]; 25 pmol of eachprimer, 200 μM of each dNTP, 1.0 mM MgCl₂ and 0.75 U of Taq polymerase(Promega, Madison, Wis.) In 200 μl PCR tube strips. The primers usedare: 5′-ACC AGG CCC CTC CAC CGG-3′ upstream (primer 9) and 5′-TCT GGTAGG GGA GCC TCA GC-3′ downstream (primer 10). The tube rack is brieflycentrifuged in a table top centrifuge and placed in a Perkin Elmer®model 9600 thermocycler programmed to run 4 minutes at 94° C.denaturation; 35 cycles of 1 minute at 94° C.; 1 minute at 58° C., 1minute at 72° C.; with a final extension of 4 minutes at 72° C. program.

[0154] Amplification 2. Two mass mixes are made for each sample. Thewild-type mass mix contains primers 9 and 11 (primer 11 is 5′-CCA CCAGGC CCC CT-3′) and the mutant mass mix contains primers 9 and 12 (5′-GCACCA GGC CCC GT-3′). With both mass mixes, 3 μl of product from the firstamplification is added to 47 μl of PCR reaction mix containing 1×PCRbuffer B (Promega; Madison, Wis.) [50 mM KCI, 10 mM Tris-HCl (pH 9.0),1.0% Triton X-100]; 25 pmol of each primer, 200 FM of each dNTP, 1.0 mMMgCI₂ and 0.75 U of Taq polymerase (Promega; Madison, Wis.) In 200 μlPCR tube strips. The tube rack is briefly centrifuged in a tabletopcentrifuge and placed in a Perkin Elmer® model 9600 thermocycler whichis programmed to run the 4 minutes at 94° C. denaturation; 35 cycles of1 minute at 94° C.; 1 minute at 52° C.; 1 minute at 72° C.; with a finalextension of 4 minutes at 72° C. programmed.

[0155] Gel Electrophoresis. The samples are electrophoresed in pairs(wild type and mutant) on a 2.0% agarose gel. Wild-type allele producesa 516 bp product only with the wild type reaction mix. The ^(*)10 mutantallele produces a 516 bp product only with the mutant reaction mix. Aheterozygote produces product with both mixes.

[0156] Conventional CYP2C9 144 Assay, Amplification. Five μl of DNA (60ng/μl) is added to 20 μl of a PCR reaction mix containing 1×PCR buffer B(Promega; Madison, Wis.) [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.0%Triton X-100]; 25 pmol of each primer, 200 μtM of each dNTP, 1.0 mMMgCl₂ and 0.75 U of Taq polymerase (Promega; Madison, Wis.) In 500 μlPCR reaction tubes. Twenty five μl of mineral oil is added to the top ofeach of the reaction tubes. The primers used are those of Bhasker etal., Pharmacogenetics 7: 51-8 (1997); 5′-TTC TCA AAA GTC TAT GGT-3′upstream and 5′-GCC TTG TGG AGG AGT TGA-3′ downstream. The tubes arebriefly centrifuged in a microcentrifuge and placed in a Perkin Elmert ®model 480 thermocycler. The amplification program used is 2 minutes at94° C. denaturation; 35 cycles of 1 minute at 94° C.; 1 minute at 50°C.; 1 minute at 72° C.; with a final extension of 4 minutes at 72° C.

[0157] Restriction Enzyme Digestion. A mass mix containing 2 μl of AvaII (New England Biolabs, NEB; Beverly, Mass.) and 3 μl of NEB Buffer, 4per reaction, is prepared, 5 μl is added to each reaction tube. Thetubes are centrifuged briefly in a microcentrifuge to bring therestriction enzyme mix below the mineral oil layer and incubated at 37°C. for at four hours.

[0158] Gel Electrophoresis. The samples are electrophoresed on a 2.0%agarose gel. Wild-type allele produces 44 and 256 base pair fragments.The 144 mutant produces a 349 base pair (bp) fragment; and aheterozygote shows 44, 256 and 349 bp bands on the gel.

[0159] Conventional CYP2C9 358 Assay.

[0160] Amplification. Five μl of DNA (60 ng/μl) is added to 20 μl of aPCR reaction mix containing 1×PCR buffer B (Promega; Madison, Wis.) [50mM KCI, 10 mM Tris-HCl (pH 9.0), 1.0% Triton X-100]; 25 pmol of eachprimer, 200 μM of each dNTP, 1.5 mM MgCl₂ and 0.75 U of Taq polymerase(Promega; Madison, Wis.) In 500 μl PCR reaction tubes, 25 μl of mineraloil is added to the top of each of the reaction tubes. The primers usedare those of Bhasker et al. Pharmacogenetics 7: 51-8 (1997); 5′-GTC CAGGAA GAG ATT GAT C-3′ upstream and 5′-CAG AAA CTA CCT CAT CCC CAA-3′downstream. The tubes are briefly centrifuged in a microcentrifuge andplaced in a Perkin-Elmer model 480 thermocycler. The amplificationprogram used is 2 min. at 94° C. denaturation; 35 cycles of 1 min. at94° C.; 1 min. at 50° C.; 1 min. at 72° C.; with a final extension of 4min. at 72° C.

[0161] Restriction Enzyme Digestion. A mass mix containing 2 μl of Nsi I(NEB; Beverly, Mass.) and 3 μl of NEB Nsi I Buffer per reaction isprepared, and 5 μl added to each reaction tube. The tubes arecentrifuged briefly in a microcentrifuge to bring the restriction enzymemix below the mineral oil layer and incubated for 4 hrs. at 37° C.

[0162] Gel Electrophoresis. The samples are electrophoresed on a 2.0%agarose gel. Wild-type allele produces a 181 bp fragment. The 358 mutantproduces 73 and 108 bp fragments. A heterozygote appears with 73, 108and 181 bp bands on the gel.

EXAMPLE 2 In vitro Model to Study Tamoxifen

[0163] I_(Kr) is one of the major polarizing currents and its block hasbeen implicated in TdP (Carlsson et al., J. Cardiovasc. Pharmacol. 16:276-85 (1990); Roden et al., Am. Hear. J. 111: 1088-93 (1986); Woosley,Annu. Rev. Pharmacol. Toxicol. 36: 233-52 (1996); and Follmer et al.,Circulation 82: 289-93 (1990)). To evaluate whether tamoxifen affectsI_(Kr), we first establish the presence of I_(Kr) in the chosen model,rabbit ventricular myocytes, using a drug known to be specific forblocker of I_(Kr). Other suitable models, such as feline myocytes orHEK293 cells expressing HERG, can be substituted for rabbit myocytes.Figure 1A and 1B show the membrane currents elicited by a 1.5-secondvoltage-clamp step from −40 mV to different test potentials ranging from−10 to −50 mV in the same cell before (Panel A) and after (Panel B) 5minutes exposure to 5 μmol/L E-4031, a highly selective I_(Kr) blocker(Clay et al., Biophys J. 69: 1830-7 (1995); and Sanguinetti et al., J.Gen. Physiol. 961: 195-215 (1990)). Under control conditions, arelatively slowly activating outward current flowed duringdepolarization, followed by an outward tail current that has been shownto represent the gradual decay of I_(Kr) (Follmer et al., Circulation82: 289-93 (1990); Clay et al., (1995); and Sanguinetti et al., (1990)).The initial peak in the time-dependent outward current was due to therapid activation and inactivation of I_(to), which is sensitive to4-aminopyridine. E-4031 abolished the tail current upon repolarizationand also reduced the time-dependent outward current, without affectingthe initial peak (I_(to)) or the holding current (I_(K1))- Shown inPanel B are the E-4031 sensitive currents obtained by digitalsubtraction of currents in the bottom tracings from currents in the toptracings in panel A. Compared with the tail current, the time- dependentcurrent demonstrated marked inward rectification at very positivepotentials, I_(to) was not present in the E-4031 sensitive currents,indicating E-4031 has no effect on I_(to) at this concentration.Superfusion with 1-2.5 μmol/L dofetilide or removal of extracellular K⁺also abolished the tail current (Liu et al., J. Pharmacol. Exp. Ther.287: 877-83 (1998)). These features of the delayed rectifier current(inward rectification of the time-dependent current, complete block ofthe tail current by E-4031, dofetilide and removal of the extracellularK⁺) are consistent with the previous description of I_(Kr) in rabbit andother species (Clay et al., (1995); and Sanguinetti et al., (1990)).

[0164] In the same cell shown in FIGS. 1A and 1B, I_(Kr), current wasalso measured before and after E-4031 exposure. FIG. 1C demonstrates theI_(K1) current elicited by a 250 ms hyperpolarization to −120 mV from aholding potential of −40 mV before and after E-4031 superfusion. Littleeffect was observed on the I_(K1) inward current, although I_(Kr) wascompletely blocked in the same cell. The outward holding currents thatrepresent the amplitude of I_(Kr) at −40 mV before and after E-4031superfusion were superimposable, indicating that E-4031 had no effect onthe I_(K1) outward current.

[0165] Effect of Tamoxifen on I^(Kr), I_(to) and I_(K1). The effect oftamoxifen on the three major potassium currents was tested using thesame protocol as described for the experiment in FIG. 1. Shown in FIG. 2are the I_(Kr), I_(to) and I_(K1) currents elicited in the same cellbefore and after 5 minutes exposure to 10 μmol/L tamoxifen. Similar toE-4031, tamoxifen abolished the I_(Kr) tail current and also reduced thetime-dependent current without affecting I_(to) or the holding current(FIG. 2A). The solvent for tamoxifen, ethanol, had no effect on I_(Kr)at the concentration (£0.1% v/v) used to dissolve tamoxifen. FIG. 2Bdepicts the tamoxifen sensitive currents obtained by digital subtractionof currents in the bottom tracings from currents in the top tracings inpanel A. There is a striking similarity between the tamoxifen sensitivecurrent and the E-4031 sensitive current shown in FIG. 1B. In both FIG.1B and 2B, the time-dependent current demonstrated strong inwardrectification at very positive potentials compared with the tailcurrent, while I_(to) was not detectable in either the tamoxifen or theE-403 1 sensitive current. FIG. 2C shows the I_(K1) current measuredbefore and after 5 minutes exposure to 10 μmol/L tamoxifen in the samecell shown in FIGS. 2A and 2B. Like E-4031, tamoxifen produced noinhibition of the I_(K1) inward current at −120 mV. In fact, I_(K1)inward current was slightly larger after tamoxifen treatment of thiscell (FIG. 2C). The outward holding currents representing the amplitudeof I_(K1) at −40 mV were superimposable, indicating that tamoxifen hadno effect on the I_(Kr) outward current.

[0166] Time-dependent Block of I_(Kr) by Tamoxifen. FIG. 3 depicts atypical experiment performed in the same cell before drug administration(control), or after 3, 5 and 9 min. superfusion with 1 μmol/L tamoxifen.As shown in FIG. 3, I_(Kr) block by tamoxifen is time-dependent and hasa slow onset. Further block can still be observed after superfusion for5 minutes. In contrast, I_(Kr) was readily recorded from controlmyocytes (no exposure to tamoxifen) for at least ten minutes without anysign of run-down. In the absence of drug, the amplitude of I_(Kr)measured ten minutes after membrane rupture was 103.4±6.15% of thatmeasured immediately after membrane rupture (n=3, p>0.05).

[0167] Effect of Tamoxifen on the I-V Relationship of I_(Kr) FIGS. 4Aand 4B demonstrate the effects of 1 and 3.3 μmol/L on the I-V(current-voltage) relationship of I_(Kr), measured after 5-7 min.infusion of tamoxifen. Tamoxifen markedly reduced I_(Kr) currentamplitude in a concentration-dependent fashion. A typicalvoltage-dependent block of I_(Kr) is shown in FIG. 4. Note the greaterblock at more positive potentials. At the test potential of +50 mV,tamoxifen (1 and 3.3 μmol/L) blocked I_(Kr) by 39.5%±1.7% (p<0.01) and84.8%±1.3% respectively (p<0.01). No significant block of I_(K1) wasobserved at 3.3 μmol/L (5.5%±0.9% test potential=−120 mV, n=4, p>0.05).

EXAMPLE 3 Comparison of I_(Kr) Block by Tamoxifen and Quinidine

[0168] Quinidine is a drug that has been frequently associated with TdP(Roden et al., Am. Heart J. 111: 1088-93 (1986)). To compare the blockof I_(Kr) by tamoxifen to that produced by quinidine, we performed theprotocol as discussed above, using either 10 μmol/L tamoxifen or 10μmol/L quinidine. Tamoxifen completely blocked the I_(Kr) tail currentwith no recovery observed after 5 min. washout, whereas the sameconcentration of quinidine (10 μmol/L) only partially blocked the I_(Kr)tail currents, with recovery within 3 min. washout. In otherexperiments, complete recovery of I_(Kr) from quinidine block wasusually observed after −5 minutes washout, while no recovery fromtamoxifen could be detected even after 15 minutes washout. FIG. 5Ccompares the percentage inhibition of I_(Kr) by tamoxifen and quinidineat the same concentration of 3.3 μmol/L. These data show that, at theleast potential of −50 mV, tamoxifen produced significantly greaterinhibition of I_(Kr) compared to quinidine (84.8%±1.3% versus42.5%±9.1%, p<0.01). Thus, tamoxifen was a more potent and longerlasting blocker of I_(Kr) than quinidine.

[0169] Effect of tamoxifen on APD and I_(Ca). We next examined whethertamoxifen causes prolongation of action potential duration (APD). FIG. 6shows action potentials recorded before and after 4 min. exposure of 3.3μmol/L tamoxifen. Surprisingly, although tamoxifen inhibited I_(Kr) byabout 84.8% at this concentration, no significant prolongation of APDwas observed. APD measured at 90% repolarization (APD₉₀) before andafter about 4-5 min. superfusion of tamoxifen (3.3 μmol/L) was 341±49 msand 332±19 ms respectively (n=16, p>0.05). Since under controlconditions, no significant shortening of APD was observed in the initial10 min. after cell membrane rupture, the absence of APD prolongation bytamoxifen was not secondary to a “rundown” phenomenon.

[0170] This unexpected effect on APD and previous reports of I_(Ca)blockade by tamoxifen (Song et al., J Pharmacol. Exp. Ther. 277: 1444-53(1996)), prompted us to study the possible effect of tamoxifen on thecardiac inward I_(Ca). Consistent with earlier studies by Song et al.(1996) in vascular smooth muscle cells, we also observed a potent effectof tamoxifen in cardiac rabbit ventricular myocytes. Significantinhibition Of I_(Ca) was observed at tamoxifen concentrations greaterthan 1 μmol/L, with almost complete inhibition observed at 10 μmol/L(FIG. 7).

[0171] Since blocking of I_(Ca) will lead to a shortening of the APD,this effect may largely cancel the I_(Kr) blocking effect of tamoxifen,which would otherwise lead to a prolongation of APD in single rabbitcardiomyocyte and prolongation of QT interval in whole heart. Theobvious discrepancy between the obviousness in rabbit (e.g., no effecton APD) and the observations in humans (e.g., QT prolongation) mayresult from different relative contributions of I_(Kr) or I_(Ca) to theAPD and/or different relative potencies of tamoxifen in blocking I_(Kr)versus I_(Ca) in difference species. The net effect of tamoxifen, aswell as other agents, on the APD in a certain species would thereforedepend on both the relative contribution of the I_(Kr) versus I_(Ca) tothe APD and the relative potency of tamoxifen in blocking I_(Kr) versusI_(Ca).

[0172] Nevertheless, the major finding from these experiments (Examples2 and 3) is that tamoxifen potently blocks the rapid component of thedelayed rectifier current, I_(Kr), in a voltage-, concentration- andtime-dependent fashion. No significant effect of tamoxifen was observedon I_(Kr) or I_(to) to at concentrations up to 10 μmol/L (FIG. 2).Tamoxifen blocks I_(Kr) with a potency even greater than quinidine, adrug that has been shown to block I_(K) and is associated with a highincidence of drug-induced TdP (Roden et al., (1986)). This is, to ourknowledge, the first study showing that tamoxifen is a potent andrelatively selective blocker.

EXAMPLE 4 ECG Arrhythmia Assessment of Ibutilide

[0173] Highly accurate and reproducible measures of the QT interval inhumans have been developed (Woosley et al., Am. J. Cardiol. 72: 36B-43B(1993); Sale et al., Clin. Pharmacol. Ther. 56: 295-301(1994)). ECGanalysis is one method of assessing arrhythmias, such as prolonged QTintervals. Ibutilide or any other pharmaceutical agent to be studied,can be assessed using an ECG. It is preferable to administer the drug(e.g., ibutilide) intravenously. Ibutilide produces a reliableprolongation of the QT interval, and its effects dissipate rapidly.Twenty male and twenty female healthy volunteers (ages 21-40) received asingle, low dose of ibutilide (0.003 mg/kg), and serial ECGs wereobtained for periods before ibutilide administration (time=0), to time=4hours (e.g., 0.5, 10, 15, 20, 30, 40, 50, 60, 90, 120, 240 and 360minutes after administration). All ECGs were 12 lead and were recordedon a computer disc and on paper at a speed of 50 mm/sec, with thesubject in a stationary, resting, supine position. ECGs were coded,randomized and blindly measured using a computer-operator interactiveprogram, employing a validated method as previously described in((Woosley et al., (1993); Sale et al., (1994)). Women were studied ateach phase of the menstrual cycle (e.g., menses, ovulation and luteal),guided by luteinising hormone (LH) surge and confirmed with estradioland progesterone plasma determinations. Men were studied only once. Themaximum and average QT changes, after each dose of ibutilide, werecompared to baseline.

[0174] The baseline QTc of females during the menses, ovulation, andluteal phases were similar (410±15 msec, 408±15 msec, and 411±12 msec,respectfully), but, as expected, they were significantly longer than thebaseline QTc in men (397±22 msec, p<0.01) (FIG. 8). To better analyzethe QTc changes over time, after the ibutilide infusion, we compared thearea under the curve of the change in QTc versus time over the 60minutes after the infusion (FIG. 9); this is the period of time whereboth maximum therapeutic and toxic effects of the drug are expected. Themean QTc change over time was significantly smaller in men than in women(p<0.05). During the first hour, women in the luteal phase of theirmenstrual cycle had the least QTc prolongation secondary to ibutilide,compared to the other two phases (ANOVA p<0.05) (FIG. 9).

[0175] In the women, the maximal changes in QTc interval after theibutilide infusion at any time interval during the menstrual, ovulationand luteal phases were 63±13, 59±17, 53±14 msec, respectively (p=ns);and in men it was 54±28 msec (p=ns). As can be seen, the QTc prolongingeffect of ibutilide was prompt and was almost dissipated by the end oftwo hours. The mean QTc prolongation over time (AUC) after ibutilide wassignificantly lower for women during the luteal phase and for mencompared to women during the other two phases of the menstrual cycle(p<0.05).

EXAMPLE 5 Role of Delayed Rectifier Potassium Current in SpontaneouslyBeating Cardiomyocytes

[0176] Delayed rectifier potassium channels are important components ofcardiac repolarization. There are two major delayed rectifier potassiumcurrents, I_(Kr) (rapid component) and I_(Ks) (slow component). Drugsthat block these currents, particularly I_(Kr), slow cardiacrepolarization and increase the risk of developing potentially fatalcardiac arrhythmias such as Torsades de Pointes. Moreover, mutations inthe genes encoding for delayed rectifier potassium channel proteins havebeen linked to long QT syndrome, a condition seen in patients at highrisk for developing Torsades de Pointes cardiac arrhythmias and suddendeath. While several specific inhibitors of I_(Kr) (d-sotalol,dofetilide, E-4031) have been available for several years, studies ofendogenous I_(Ks) have been hampered by a lack of pharmacological toolsto selectively block its activity. Recently, however, a new compound,chromanol 293B, has been reported to be a relatively selective blockerof I_(Ks) (Busch et al., Pflug. Arch. 432: 1094-6 (1996)). In thepresent report, we have tested this compound using spontaneously beatingcultures of neonatal rat cardiomyocytes.

[0177] In addition to the different electrophysiological properties ofI_(Kr) versus I_(Ks) that have been described by others, these currentscan be distinguished pharmacologically (Sanguinetti et al., J. Gen.Physiol. 96: 195-215 (1990)). For example, E-4031 (Eisai Co., Ltd.,Ibaraki, Japan) has been reported to selectively block I_(Kr) atconcentrations up to 5-10 mM (Sanguinetti et al., (1990)), and chromanol293B (Hoechst Marion Roussel, Frankfurt, Germany) appears to selectivelyblock I_(Ks) at concentrations up to 10-30 mM (Busch et al., (1996)). Todetermine if pharmacological blockade of delayed rectifier potassiumcurrents influences the spontaneous beating rate of cultured neonatalrat cardiomyocytes, a dose-response curve was generated for chromanol293B. As shown in FIG. 1, chromanol 293B caused a dose-dependentdecrease in beating rate, but even at the highest drug concentrationtested (100 mM), the reduction in beating rate was only about 50% thatof control. In guinea pig ventricular myocytes, the IC₅₀ value for 293Binhibition of I_(Ks) is approximately 2 mM, although concentrations ashigh as 100 mM were required to achieve complete blockade of I_(Ks) inthose cells (Busch et al., (1996)).

[0178] We observed a similar type of dose-response relationship with theI_(Kr)-blocking compound, E-4031; however, the most interesting resultswere observed when E-4031 and 293B were added together (Panel B). Whilehigh concentrations (5-10 mnM E-4031 and 50-100 mM 293B) of either drugalone were insufficient to completely block beating activity (maximuminhibition =50%), the combination of these two compounds totally blockedall beating activity. Remarkably, treatment with the β-adrenergicagonist, isoproterenol (0.1-1 mM), allowed for the recovery of most ofthe beating activity (70-80% of control values) despite the continuedpresence of the I_(Kr) and I_(Ks) inhibitors.

[0179] The simplest explanation for these results is that there may be afunctional redundancy between I_(Kr) and I_(Ks), and complete inhibitionof either current alone will cause some slowing of beating rate due toloss of repolarization capability. However, when I_(Kr) and I_(Ks) areboth completely blocked, the cells cannot repolarize sufficiently toallow the next depolarization to occur, and hence, they stop beating.Isoproterenol appears to be able to largely overcome this inhibition,probably through its well-known stimulatory effects on calcium channels(increasing availability of I_(Ca) at more positive voltages) andperhaps through direct stimulation of I_(Ks) as well (Yazawa et al., J.Physiol. 421: 135-150 (1990); Pignier et al., Journal of CardiovascularPharmacology 31: 262-270 (1990); and Iijima et al., J. Pharmacol. Exp.Ther. 254: 142-146 (1990)).

[0180] All cited patents and publications referred to in thisapplication are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method for determining whether a subject has apredisposition for QT interval elongation when treated with one or morepharmaceutical agents comprising the step of: (A) screening a biologicalsample from the subject through a nucleic acid array, wherein saidnucleic acid array contains probes for a genetic mutation orpolymorphism in at least two or more genes, wherein the genes areselected from two or more of the categories of genes: (1) LQT genes, (2)altered sensitivity genes or (3) increased exposure genes.
 2. The methodof claim 1, wherein the gene classes are selected from the groupconsisting of (1) LQT genes, (2) altered sensitivity genes, and (3)increased exposure genes.
 3. The method of claim 1, wherein theincreased exposure gene is selected from the group consisting ofmulti-drug resistance (MDR) genes or cytochrome P450 genes.
 4. Themethod of claim 1, wherein the nucleic acid array is a chip, a bead or amicrosphere, or a microchip.
 5. The method of claim 1, wherein the LQTgene is selected from the group consisting of: LQT1, LQT2, LQT3, LQT4,LQT5 and LQT6.
 6. The method of claim 3, wherein the P450 cytochromeisoform is selected from the group consisting of: CYP1A2, CYP2C19,CYP2C9, CYP2D6, CYP2E1, CYP3A, CYP3A4, CYP3A5 and CYP3A7.
 7. The methodof claim 1, wherein the ion channel gene is a LQT gene and the geneticmutation in the LQT gene is selected from the group consisting of: HERG(nt 1778, point mutation), KCNE1 (nt 226, point mutation), KCNQ1 (nt217, point mutation), KCNQ1 (nt 284, point mutation), KCNQ1 (nt 475,point mutation), KCNQ1 (nt 521, point mutation), KCNQ1 (nt 629, pointmutation), KCNQ1 (nt 655, point mutation), KCNQ1 (nt 659, pointmutation), KCNQ1 (nt 737, point mutation), KCNQ1 (nt 746, pointmutation), KCNQ1 (nt 1378, point mutation), KCNQ1 (nt 1244-1250,deletion-insertion) and SCN5A (nt 4513-4521, 9 bp deletion).
 8. Themethod of claim 1, wherein the pharmaceutical agent is selected from thegroup consisting of: Amiodarone, Amitriptyline, Amoxapine, Astemizole,Azelastine, Bepridil, Chlorpromazine, Cisapride, Clarithromycin,Clemastine, Clomipramine, Desipramine, Diphenhydramine, Disopyramide,Doxepin, Erythromycin, Felbamate, Flecainide, Fluconazole,Fludrocortisone, Fluoxetine, Fluphenazine, Fluvoxamine, Foscarnet,Fosphenytoin, Halofantrine, Haloperidol, Thutilide, Imipramine,Indapamide, Ipecac, Isradipine, Itraconazole, Ketoconazole,Levomethadyl, Maprotiline, Moexipril/HCTZ, Moricizine, Naratriptan,Nicardipine, Nortriptyline, Octreotide, Pentamidine, Perphenazine,Pimozide, Probucol, Procainamide, Prochlorperazine, Protriptyline,Quetiapine, Quinidine, Risperidone, Salmeterol, Sotalol, Sparfloxacin,Sumatriptan, Tamoxifen, Terfenadine, Thioridazine, Thiothixene,Tizanidine, Tocainide, Trifluoperazine, Trimethoprim Sulfamethoxazole,Venlafaxine and Zolmitriptan.
 9. A method for determining whether asubject has a predisposition for QT interval elongation when treatedwith one or more pharmaceutical agents comprising the step of: (A)screening a biological sample from the subject through a DNA array,wherein said DNA array contains probes for at least two geneticmutations or polymorphisms, and wherein at least one mutation orpolymorphism is located on a LQT gene and at least a second mutation orpolymorphism is located in at least one of classes of genes selectedfrom the group consisting of: altered sensitivity genes and increasedexposure genes.
 10. The method of claim 9, wherein the LQT gene isselected from the group consisting of: LQT1, LQT2, LQT3, LQT4, LQT5 andLQT6.
 11. The method of claim 9, wherein the increased exposure gene isselected from the group consisting of: cytochrome P450 genes and MDRgenes.
 12. The method of claim 11, wherein the cytochrome P450 gene isselected from the group consisting of: CYP1A2, CYP2C19, CYP2C9, CYP2D6,CYP2E1, CYP3A, CYP3A4, CYP3A5 and CYP3A7.
 13. The method of claim 9,wherein the altered sensitivity gene is selected from MiRP1 or relatedgenes.
 14. The method of claim 9, wherein the DNA array is a chip, abead or a microsphere, or a microchip.
 15. The method of claim 9,wherein a LQT mutation or polymorphism is selected from the groupconsisting of. HERG (nt 1778, point mutation), KCNE1 (nt 226, pointmutation), KCNQ1 (nt 217, point mutation), KCNQ1 (nt 284, pointmutation), KCNQ1 (nt 475, point mutation), KCNQ1 (nt 521, pointmutation), KCNQ1 (nt 629, point mutation), KCNQ1 (nt 655, pointmutation), KCNQ1 (nt 659, point mutation), KCNQ1 (nt 737, pointmutation), KCNQ1 (nt 746, point mutation), KCNQ1 (nt 1378, pointmutation), KCNQ1 (nt 1244-1250, deletion-insertion) and SCN5A (nt4513-4521, 9 bp deletion).
 16. A nucleic acid array comprising nucleicacids which recognize and bind to at least two gene mutations orpolymorphisms located in genes found in at least two classes of genesselected from the group consisting of: (1) LQT genes, (2) alteredsensitivity genes and (3) increased exposure genes.
 17. The nucleic acidarray of claim 16, wherein the altered sensitivity gene is MiRP1. 18.The nucleic acid array of claim 16, wherein the increased exposure geneis selected from the group consisting of MDR genes and cytochrome P450genes.
 19. The nucleic acid array of claim 18, wherein the cytochromeP450 gene is selected from the group consisting of: CYP1A2, CYP2C19,CYP2C9, CYP2D6, CYP2E1, CYP3A, CYP3A4, CYP3A5 and CYP3A7.
 20. The DNAarray of claim 16, wherein the LQT gene is selected from the groupconsisting of LQT1, LQT2, LQT3, LQT4, LQT5 and LQT6.
 21. The nucleicacid array of claim 16, wherein the array comprises a nucleic acid whichdetects at least one of the following LQT gene mutations orpolymorphisms: HERG (nt 1778, point mutation), KCNE1 (nt 226, pointmutation), KCNQ1 (nt 217, point mutation), KCNQ1 (nt 284, pointmutation), KCNQi (nt 475, point mutation), KCNQ1 (nt 521, pointmutation), KCNQ1 (nt 629, point mutation), KCNQ1 (nt 655, pointmutation), KCNQ1 (nt 659, point mutation), KCNQ1 (nt 737, pointmutation), KCNQ1 (nt 746, point mutation), KCNQ1 (nt 1378, pointmutation), KCNQ1 (nt 1244-1250, deletion-insertion) and SCN5A (nt4513-4521, 9 bp deletion).
 22. A nucleic acid array comprising nucleicacids which recognize and bind to mutations and/or polymorphisms of LQTgenes, altered sensitivity genes or increased exposure genes.
 23. Thenucleic acid array of claim 22, wherein the LQT genes are LQT1, LQT2,LQT3, LQT4, LQT5, orLQT6.
 24. The nucleic acid array of claim 22,wherein the altered sensitivity gene is MiRP1.
 25. The nucleic acidarray of claim 22, wherein the increased exposure genes are MDR genes orcytochrome P450 genes.
 26. The nucleic acid array of claim 16, whereinthe array is a chip, a microchip, a microsphere or a DNA bead array. 27.A nucleic acid array which is capable of screening the 22 alleles of theCYP2D6 gene and further comprises a nucleic acid which recognizes andbinds to at least one ion channel gene mutation.
 28. The nucleic acidarray of claim 27, wherein the ion channel gene mutation is a LQT genemutation or a MDR gene mutation.
 29. A method of screening apharmaceutical agent in vitro for its ability to induce prolongedcardiac repolarization in a cell comprising the steps of: (A) measuringI_(Kr) and I_(Ks) currents of the cell using a voltage clamp beforesuperfusing the cell with a candidate agent or agents; (B) superfusingand incubating the cell with the candidate agent or agents; (C)measuring the I_(Kr) and I_(Ks) currents after superfusion andincubation of the cell with the candidate agent or agents using avoltage clamp; and (D) determining whether both the I_(Kr) or the I_(Ks)current is inhibited or abolished thereby indicating that the drugprolongs repolarization.
 30. The method of claim 29, further comprisingthe step of determining whether I_(to) is inhibited by also measuringI_(to) at each of the steps that I_(Kr) and I_(Ks) are measured.
 31. Themethod of claim 29, wherein the cell is a rabbit cardiomyocyte, a felinecardiomyocyte or HEK293 cells expressing HERG.
 32. A method of screeningtwo or more pharmaceutical agents in vitro for their ability to inducecardiac arrhythmias associated with QT interval elongation comprisingthe steps of: (A) measuring I_(Kr) and I_(Ks) currents of a cell using avoltage clamp before superfusing the cell with the two or morepharmaceutical agents; (B) superfusing and incubating the cell with thetwo or more pharmaceutical agents; (C) measuring the I_(Kr) and I_(Ks)currents after superfusing and incubation of the cell with the two ormore pharmaceutical agents using a voltage clamp; (D) comparing theI_(Kr) and I_(Ks) currents of the combined pharmaceutical agents on thecell with the I_(Kr) and I_(Ks) currents of each of the pharmaceuticalagents alone in the cell; and (E) determining whether the I_(Kr) andI_(Ks) currents are both inhibited such that beating of the cells issubstantially inhibited.
 33. A method of identifying a geneticpolymorphism which can cause QT interval prolongation in a subjectcomprising the steps of: (A) inserting a nucleic acid or nucleic acidsinto a cell wherein the nucleic acid or nucleic acids encode at leasttwo polymorphisms and/or mutations of at least two genes selected fromthe group of gene consisting of (1) LQT genes, (2) altered sensitivitygenes and (3) increased exposure genes; (B) measuring I_(Ks) and I_(Kr)currents of the cell before administering a drug known to cause a changein I_(Ks) and/or I_(Kr); (C) measuring I_(Ks) and I_(Kr) currents of thecell after superfusion of the cell with the drug; (D) comparing theI_(Ks) and I_(Kr) values of the cell expressing the polymorphisms and/ormutations to the I_(Ks) and I_(Kr) value of a cell expressing wild-typegenes; and (E) determining if the presence of the mutations and/orpolymorphisms leads to a greater inhibition or blockage of I_(Ks) and/orI_(Kr).
 34. The method of claim 33, wherein the altered sensitivity geneis MiRP1.
 35. The method of claim 33, wherein the LQT gene is selectedfrom the group consisting of: LQT1, LQT2, LQT3, LQT4, LQT5 and LQT6. 36.The method of claim 33, wherein the increased exposure gene is selectedfrom the group consisting of MDR genes and cytochrome P450 genes. 37.The method of identifying a genetic polymorphism of claim 36, whereinthe cytochrome P450 gene is selected from the group consisting ofCYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A, CYP3A4, CYP3A5 andCYP3A7.
 38. The method of claim 33, wherein the drug known to cause achange in I_(Ks) or I_(Kr) is selected from the group consisting ofE-4031, 293B, Ibutilide, Chromanol, Dofetilide, Tamoxifen, Cisapride,Zolmitriptan, Venlafaxine, Tizanidine, Sumatriptan, Salmeterol,Rispiradone, Fosphenytoin, Isradipine, Levomethadyl, Quetiapine,Pimozide, Azelastine, Chlorpromazine, Fluoxetine, Foscarnet,Grepafloxacin, Naratriptan and Nicardipine.